WO2001029262A2 - Genotyping reagents, kits and methods of use thereof - Google Patents

Abstract

The present invention provides oligonucleotides that can be used to determine the presence, absence or identity of a single nucleotide polymorphism (SNP), kits containing such oligonucleotides, and methods of genotyping a nucleic acid sample using such oligonucleotides.

Description

GENOTYPING REAGENTS, KITS AND METHODS OF USE THEREOF

Background

Sequencing the human genome has provided information to dramatically change the way diseases are studied, diagnosed and treated. The fields of pharmacogenomics and pharmacogenetics focus on the variability and diversity of DNA, and how this diversity can impact biology, including response to drugs. The most common form of genetic diversity is thought to come in the form of individual DNA bases being different than the corresponding base in the average population. These single nucleotide polymorphisms ("SNPs") are a natural form of mutation, which are hereditary in nature. SNPs are viewed as both direct and indirect markers for many human diseases. Therefore, the analysis, or "scoring," of SNPs to determine their role in disease and drug efficacy can lead to the development of powerful diagnostics and therapeutics.

Adverse drug reactions (ADRs) represent the leading cause of hospitalization and mortality in the

U.S., resulting in over 1.5 million hospitalizations and 100,000 deaths per year (J. Lazarou, et.al. in The Journal of the American Medical Associa tion (JAMA) , (1998, 1200-1205). In addition, many promising drugs developed by pharmaceutical companies never make it through clinical development because of genetically based adverse reactions (ADRs) in a small number of patients. Therefore, scoring SNPs can identify at risk patients before the drug is prescribed and salvage potentially useful new drugs. Alternatively, patients with SNP- induced variations in their conditions, evident in diseases like hypertension, can be identified and then prescribed the drug most appropriate for their genetic variant. Therefore, SNP analysis will be used to streamline drug discovery and development leading to safer and more effective tailor-made drugs.

Accordingly, new reagents are needed that can be used to identify whether heretofore unknown SNPs are present or absent from a given nucleic acid sample. The present invention satisfies this need and provides related advantages as well.

Summary of the Invention

In accordance with the present invention, there are provided oligonucleotides that can be used to determine the presence, absence or identity of a single nucleotide polymorphism. Invention oligonucleotides include genotyping oligonucleotides that hybridize with a portion a nucleic acid sequence listed in column 4 of Table 1, or complementary sequence thereof, and amplification oligonucleotides.

In accordance with another embodiment of the invention, there are provided kits useful for determining the presence, absence or identity of a single nucleotide polymorphism. In one embodiment, an invention kit comprises an oligonucleotide that hybridizes with a portion of a nucleic acid sequence listed in column 4 of Table 1, or complementary sequence thereof. In another embodiment, an invention kit comprises amplification oligonucleotides of columns 1 and 2 of Table 1. Also provided is an invention kit comprising an oligonucleotide that hybridizes with a portion of a nucleic acid sequence listed in column 4 of Table 1, or complementary sequence thereof and two amplification primers .

Another embodiment of the invention provides methods of genotyping a nucleic acid sample by hybridizing a genotyping oligonucleotide to a nucleic acid sequence listed in column 4 of Table 1, or complementary sequences thereof. The genotyping oligonucleotide can then be employed in a variety of reactions, such as, for example, a primer extension reaction. Also provided is a method of genotyping a nucleic acid sample by amplifying a target nucleic acid sequence and performing a single-nucleotide primer extension reaction employing a genotyping oligonucleotide that hybridizes to the target nucleic acid sequence.

Brief Description Of The Figures

Figure 1 shows a schematic for genotyping a heterozygous nucleic acid sample containing two target nucleic acids using the particular primer extension reaction referred to as Single Nucleotide Primer Extension (SNPE) and/or GBA. In step 1, genomic DNA is amplified using a pair of PCR primers from columns 1 and 2 of the same row of Table 1 herein, in which the five most 5' nucleotides of one of the two PCR primers are linked by four exonuclease-resistant phosphorothioated linkages (e.g., 5' C- (p) -G- (p) -C- (p) -A- (p) -GTCTCAGGCCAGCT 3', for SEQ ID NO: 2, where "-(p)-" represents a phosphorothioate linkage) . The PCR primers are complementary to sequences flanking either side of a specific nucleotide site (e.g., the site of a SNP) in the genomic DNA, thus resulting in an amplified nucleic acid (the target nucleic acid) containing this specific site. In step 2, the amplified PCR product target nucleic acid is subjected to T7 gene 6 exonuclease digestion, which removes the strand of the target nucleic acid that did not include the 5' phosphorothioated linkages, resulting in a single-stranded target nucleic acid. In step 3, the genotyping primer (or SNPE primer) is hybridized with the single stranded target nucleic acid such that the specific nucleotide of the target nucleic acid that is to be analyzed (i.e., the SNP) is not hybridized, and is immediately adjacent the 3' end of the genotyping primer. In step 4, a terminator reagent mixture comprising dideoxy terminators corresponding to A, C, G and T, together with a polymerase, are added to the target nucleic acid-genotyping primer complex. The polymerase extends the genotyping primer by a single base, consisting of one of the four terminators, where the added terminator base is complementary to the SNP to be determined. The terminator base that is added to the genotyping primer is labeled with a detectable marker. In the second part of step four, NaOH is added to dissociate the target nucleic acid strand which is washed away, while the genotyping primer, fixed to a solid support, remains. In step 5, a detection reagent is added, if necessary, to generate signal from the detectable marker. This can be performed in an ELISA format where an enzyme-linked antibody specific for the detectable marker of a terminator is contacted with the extended genotyping primer. When detectable markers that can be distinguished from each other are used, multiple alleles can be identified in a single well. For example, as illustrated in step 5, two alleles can be identified (C and T) by the sequential use of antibodies selective for each of the terminators (ddCTP and ddTTP) . Figures 2A and 2B show schematics for genotyping a homozygous nucleic acid sample using the Single Nucleotide Primer Extension (SNPE) by the enzyme- linked immunosorbant assay (ELISA) detection method after amplification using a phosphorothioated primer and exonuclease digestion. In figure 2A, the template strand indicated in the figure is a representative target nucleic acid molecule (such as those listed in SEQ ID NO:4n+4) . The SNPE primer indicated in the figure is a representative genotyping primer (such as those listed in SEQ ID NO:4n+3). The nucleotide to be interrogated is indicated as the SNP to be typed. Primer extension is performed as described herein, in which the polymerase extension step can be performed in the presence of the terminators ddATP, ddGTP, fluorescein-ddCTP and biotin- ddUTP. The last two terminators are linked to detectable markers. Figure 2B shows the detection step after the single nucleotide primer extension results in the addition of fluorescein-ddCTP onto the 3 ' -end of the SNPE primer. The diagram illustrates the interaction of alkaline phosphatase-conjugated anti-fluorescein antibody and the generation of signal at OD₄₀₅ after the addition of PNPP (p-nitrophenyl phosphate) substrate. Since the biotin-ddUTP is not incorporated, the anti-biotin horse radish peroxidase (HRP)does not bind to the primer, no reaction with the TMB (tetramethylbenzidine) substrate occurs and, hence, no signal is generated at OD₆₂₀.

Figure 3 shows a scatterplot of OD₄₀₅ (X-axis) versus OD₆₂₀ (Y-axis) resulting from SNPE that demonstrates a result that is heterozygous. The PCR primers used were oligonucleotides of SEQ ID NOs:25 and 26. The SNPE primer used was an oligonucleotide with SEQ ID NO:27. The targeted nucleic acid with the suspected polymorphism contains the sequence of SEQ ID NO: 28. SNPE was performed using fluorescein-ddCTP and biotin-ddUTP . The Y-axis corresponds to signal obtained from ddUTP incorporation and the X-axis corresponds to signal obtained from ddCTP incorporation. Each circle (•) indicates a result with a specific test sample (unknown) from an individual genomic DNA. Plus (+) symbols are positive controls using a synthetic template. Crosses (x) are negative controls where no PCR is performed.

Detailed Description of the Invention

In accordance with the present invention, there are provided oligonucleotides that can be used to determine (genotype) the presence, absence or identity of a single nucleotide polymorphism (SNP) at a preselected site in a human genomic nucleic acid sample. The location of the SNP in each SNP-containing oligonucleotide set forth in column 4 of Table 1 (also referred to herein as SEQ ID NO:4n+4, wherein n=0 through 934) herein corresponds to the nucleotide position on each SEQ ID NO:4n+4 that is immediately adjacent the nucleotide complementary to the 3' end of the corresponding genotyping oligonucleotide set forth in column 3 of the same row of Table 1. The nucleotide set forth on SEQ ID NO:4n+4 corresponding to the SNP site corresponds to the predominant polymorphism.

As set forth in Table 1, each row of oligonucleotides disclosed corresponds to a set of oligos useful for genotyping a nucleic acid sample for the presence or absence, or identity of a particular SNP. The oligonucleotides set forth in columns 1 and 2 of

Table 1 (also referred to herein as SEQ ID N0:4n+1 and SEQ ID NO:4n+2, respectively, where n=0 through 934) are used as primers to amplify the nucleic acid sample having the corresponding SNP-containing oligonucleotide from the same row in Table 1 (referred to as SEQ ID NO:4n+4, where n=0 through 934) . See Figure 1. Once the nucleic acid sample having the corresponding SNP-containing oligonucleotide has been amplified, the corresponding genotyping oligonucleotide from column 3 (referred to as SEQ ID NO:4n+3, where n=0 through 934) of the same row in Table 1 is used to genotype the target nucleic acid, preferably by a primer extension reaction.

As used herein, the term "oligonucleotide", "oligo" or grammatical variations thereof, refers to polynucleic acid that can be either DNA or RNA. Invention oligos can be produced by methods well-known in art such as described in the Examples hereinafter; or the oligos can be purchased from commercial oligonucleotide production companies.

As used herein, the phrase "SEQ ID NO:4n+3, wherein n=0 through 934" refers to the group of oligonucleotides in the Sequence Listing that are set forth in column 3 of Table 1 herein (where n=(row number - 1) . For example, n=0 corresponds to SEQ ID NO: 3, n=l corresponds to SEQ ID NO:7,..., n=934 corresponds to SEQ ID NO: 3739 of the Sequence Listing. Likewise, SEQ ID NO:4n+l, SEQ ID NO:4n+2, and SEQ ID NO:4n+4, wherein n=0 through 934, refers to the groups of oligonucleotides set forth in columns 1, 2 and 4, respectively, of Table 1 herein.

The oligonucleotides set forth in column 3 of Table 1 (SEQ ID NO:4n+3, where n=0 through 934) are also referred to herein as genotyping oligonucleotides. In addition, for each SNP located in each oligonucleotide set forth in column 4 of Table, there are at least four potential genotyping oligonucleotides (e.g., genotyping primers) that are hybridizable to the region one nucleotide removed from either 5 ' or 3 ' end of the SNP nucleotide (two for each strand) . Two of the genotyping primers (one for each target nucleic acid strand) are complementary to the target nucleic acid and have their 3¹ ends one nucleotide 3' to the SNP nucleotide. These primers can be used, for example, in a polymerase primer extension reaction such as described in Goelet, P. et al., U.S. Pat No.5,888,819 and Nikiforov, T.T. et al., U.S. Pat. No. 5,518,900, which are herein incorporated by reference. The other two primers are complementary to the target nucleic acid and have their 5 ' ends one nucleotide 5' to the SNP nucleotide. These primers can be used in, for example, a ligase/polymerase genotyping reaction such as described in Nikiforov, T.T. et al . , U.S. Pat. No. 5,679,524, or a nucleotide and terminator mixture reaction such as described in Soderlund et al . , U.S. Pat. No. 6,013,431 or by Koster et al . , U.S. Pat.

No. 6,043,031, which is herein incorporated by reference.

In addition, the oligonucleotides set forth in column 3 of Table 1 (SEQ ID NO:4n+3, where n=0 through 934) can be made one nucleotide longer such that the nucleotide at the 3' end lies complementary to the SNP nucleotide. These primers can be used in alternate genotyping methods, for example, in allele-specific polymerase chain reaction (AS-PCR) (Newton et al., 1989, Nucl Acids Res 17:2503-2516), ligase chain reaction (LCR) (Barrany, 1991, PNAS, USA, 88: 189-193), or oligonucleotide ligation assay (OLA) (Landegren et al . , 1988, Science 241:1077), where the 3' nucleotide of the genotyping primer determines the presence of the SNP nucleotide . In addition, the oligonucleotides set forth in column 3 of Table 1 (SEQ ID NO:4n+3, where n=0 through 934) can be made one or more nucleotides shorter such that the genotyping primers are complementary to the target nucleic acid and have their 3 ' ends two or more nucleotides 3' to the SNP nucleotide. These primers can be used in, for example, a labeled nucleotide and terminator mixture reaction such as described in Soderlund et al . , U.S. Pat. No. 6,013,431 or by Koster et al., U.S. Pat. No. 6,043,031, which is herein incorporated by reference.

The genotyping primer can be an oligodeoxyribonucleotide (DNA), an oligoribonucleotide (RNA) , or a copolymer of deoxyribonucleotides and ribonucleotides . The genotyping primer can be either natural or synthetic. The genotyping primer can be synthesized either enzymatically in vivo, enzymatically in vitro, or non-enzymatically in vitro. The genotyping primer can include modifications of the ribose-phosphate backbone, such as phosphorothioate or methylphosphonate or other backbone modifications such as peptide nucleic acid (PNA) . The genotyping primer can also include modifications of the nucleotide bases. The genotyping primer can be labeled with a detectable marker; this detectable marker can be different from any detectable marker present in the reaction mixture or any detectable marker attached to the target nucleic acid. In addition, for use with single-nucleotide primer extension reactions, the genotyping primer must be capable of hybridizing or annealing with nucleotides present in the target nucleic acid, immediately adjacent to, and upstream of, the nucleotide base to be identified. One way to accomplish the desired hybridization is to have the template-dependent primer be substantially complementary or fully complementary to the known base sequence immediately adjacent to the base to be identified. The genotyping primer may be bound to a solid surface such as described in U.S. Pat. No. 5,610,287, incorporated herein by reference in its entirety. Multiple different genotyping primers can be immobilized on a surface to create an array, thus allowing the determination of multiple genotypes in a single reaction. Multiple different genotyping primers with distinct attachment means ("tags") can used for multiplex single nucleotide primer extension in solution and subsequently separated for discrete detection, such as on a nucleic acid array comprising nucleic acids complementary to the attachment means of the genotyping primers ("tag arrays") .

Genotyping oligonucleotides of the invention can be as long as the nucleotide distance between an amplification primer and the SNP nucleotide, preferably not overlapping the amplification primer sequence, preferably less than 90 nucleotides, preferably less than 50 nucleotides, more preferably between 14 and 50 nucleotides, most preferably between 17 and 30 nucleotides .

When invention genotyping oligonucleotides are used in, for example, single-primer-extension reactions, hairpin loop structures can form due to self- complementarity of the nucleotides sequences of the oligo/primer . In order to disrupt the hairpin loop structure formation, "single-nucleotide-spacers" can be inserted in the midst of the hairpin loop structure. As used herein, the term "single-nucleotide-spacer" refers to any non-Watson-Crick moiety incorporated within an oligonucleotide for the purpose of weakening nucleic acid duplex stability without altering the fidelity of the surrounding Watson-Crick base pairs. Thus, modifications to genotyping oligonucleotides contemplated herein are for the purpose of destabilizing duplex formation, specifically disfavoring intrastrand folding in favor of interstrand pairing. The location of single-nucleotide- spacers are set forth in the Sequence Listing and column 3 of Table 1 as an "X". Such moieties are well-known and available in the art and can include anucleosidic moieties, abasic moieties, non-naturally occurring nucleotide analogs, and non-Watson/Crick base moieties.

As used herein, the term "anucleosidic moieties" refers to moieties that approximate the spacing of the phosphoribose linkage. Thus, anucleosidic sites are contemplated herein as space-holders between neighboring phosphates along the oligo backbone. Examples of anucleosidic moieties for use in invention oligonucleotides include phosphoramidite-coupled multicarbon aliphatic chains that lack a nucleotide base and approximate the internucleotide spacing. For example, a C-3 linker used in DNA synthesis (3- (4, 4'- Dimethoxytrityloxy) propel-1-phosphoramidite) (Glen Research, Sterling, VA) will have the approximate spacing of one nucleotide within a nucleic acid chain. Other anucleosidic sites include ethyl-like C2 and Cl linkers, rather than C3. C2- and C3-linkers are preferred as single-base length analogs. Larger anucleosidic spacers are contemplated herein having stretches of a few C2 or C3 linkers, or by incorporating a single C6 linker (for a two-base gap), or C9 (for 3 bases), and so forth up C18 linkers, and the like.

As used herein, the phrase "abasic moiety" refers to moieties that approximate the conformation of a nucleotide but are chemically distinct from nucleotides, which include sugars or sugar analogs without base moieties. For example, an abasic site consists of the sugar-phosphate backbone without the base, such as tetrahydrofuran-phosphate (dSpacer; Glen Research,

Sterling, VA) , can be used as a nucleotide analog. Other backbones include those in which one or two of the non- bridging oxygen atoms of the phosphate moiety of a nucleotide have been replaced with a sulfur-containing group (especially a phosphorothioate) , an alkyl group

(especially a methyl or ethyl alkyl group) , a nitrogen- containing group (especially an a ine) , and/or a selenium-containing group. In addition, peptide nucleic acids (PNAs) (Buchardt O, Trends Biotechnol 1993 Sep; 11 ( 9) : 384-6) or a carbohydrate are alternate backbones that can be used. It is contemplated that abasic modifications of these backbone types can be used as nucleotide analogs.

As used herein, "non-natural base variations" are non-canonical bases often referred to as "degenerate bases" since they exhibit some ability to base pair to any of the 4 standard bases. Exemplary non-natural base variations include, for example, "purine" and "pyrimidine" (which would be the structural scaffolds for A/G and C/T, respectively) , as well as fluorine- derivatized bases, and the like. Examples of other non- natural base variations include 5-nitroindole, 3- nitropyrrole, and inosine. Numerous other "natural" base modifications that result from carcinogen exposure are also contemplated herein (see, e.g., Glen Research

Catalog) . The single nucleotide spacer can also include nucleotide ribose or deoxyribose analogs. As used herein, the phrase "naturally occurring, non-Watson/Crick, bases" refers to any naturally occurring canonical base other than dA, dC, dG, dT . Common bases would be doexyinosine (dl) and deoxyuridine (dU) . Other naturally occurring base variants can be found in tRNA sequences, and the like.

The single nucleotide spacer can also include non-complementary Watson/Crick nucleotide bases. An example of a mismatched nucleotide base would be the substitution of a thymidine base with an adenine base such that the intra helical base pair would be A:A instead of T:A and hence disrupt base pairing. For example, it is well known that some non-Watson/Crick pairs have moderate stability, such as G/T pairs. For any base pair, there are 6 potential mis-pairs using the standard 4 bases .

As used herein, the term "hybridize", or grammatical variations thereof, refers to the ability of two strands of nucleic acid molecules to hydrogen bond in a sequence dependent manner. For example, under appropriate conditions, complementary nucleotide sequences can hybridize to form double stranded DNA or RNA, or a double stranded hybrid of RNA and DNA. Nucleotide changes in the complementary sequence that disrupt base-pairing result in a decreased stability of the hybrid. These concepts are known in the art and described in Sambrook et al . , Molecular Cloning: A Laboratory Manual 2nd ed . (1989), which is incorporated herein by reference.

Stringency levels used to hybridize a given probe with target-DNA can be readily varied by those of skill in the art. The phrase "stringent hybridization" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T_m) of the hybrids. In general, the stability of a hybrid is a function of GC content, cation concentration, formamide concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions .

As used herein, the phrase "moderately stringent hybridization" refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the target DNA; with greater than about 90% identity to target-DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5X Denhart ' s solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 65°C.

The phrase "high stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein) . High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart ' s solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0. IX SSPE, and 0.1% SDS at 65°C. The phrase "low stringency hybridization" refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart' s solution, 6X SSPE, 0.2% SDS at 42°C, followed by washing in IX SSPE, 0.2% SDS, at 50°C. Denhart ' s solution and SSPE (see, e.g.,

Sambrook et al . , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers.

As used herein, the phrase "immediately adjacent the 3' end" refers to a specific first nucleotide position, typically on the target nucleic acid or on a nucleic acid having SEQ ID NO:4n+4, or complementary sequences thereof, which is immediately 5' of a second nucleotide position on the same nucleic acid, in which the second nucleotide position is complementary to the 3 ' -end nucleotide of SEQ ID NO:4n+3 or complementary sequence thereof. For example, Figure 2A shows a nucleotide sequence representative of a target nucleic acid sequence or a nucleic acid sequence having SEQ ID NO:4n+4, labeled "Template strand." Figure 2A also shows a nucleotide sequence representative of a nucleic acid sequence having SEQ ID NO:4n+3, labeled "SNPE primer." The 3 ' -end nucleotide of the SNPE primer is an "A" which is complementary to a "T" on the Template strand. The nucleic acid immediately 5' of the "T" on the Template strand is a "G," and is labeled in the figure "SNP to be typed. Accordingly, the "G" nucleotide on the Template strand, labeled as the SNP to be typed, is "immediately adjacent the 3' end" of the SNPE primer in Figure 2A.

Preferably, the invention oligonucleotides corresponding to SEQ ID NOs:4n+l and 4n+2, where n=0 through 934 (i.e., columns 1 and 2 of Table 1) can be used as two amplification primers for increasing the amount of target nucleic acids (set forth in column 4 of Table 1) containing the SNP. In addition, in view of the target nucleic acids disclosed herein in column 4 of Table 1, those of skill in art can readily design additional amplification primers that can amplify larger target nucleic acid fragments comprising the target nucleic acids set forth in column 4. Likewise, it is contemplated herein that those of skill in art can readily design additional amplification primers that can amplify smaller target nucleic acid fragments, so long as the smaller target nucleic acid comprises the SNP-site set forth in column 4. The amplification primer pairs are designed such that the primers generate a target nucleic acid strand complementary to the genotyping primer and resistant to degradation, for example, by 5'- 3 ' exonuclease .

As used herein, a nucleic acid that is "resistant to degradation" or "degradation-resistant" is a nucleic acid that is resistant to chemical degradation, acid hydrolysis, base hydrolysis, or other chemical- induced hydrolysis, or is resistant to enzymatic degradation. A nucleic acid that is resistant to enzymatic degradation can be resistant to exonuclease activity, endonuclease activity, and the like. In a preferred embodiment, a nucleic acid that is resistant to degradation is resistant to 5 '-3' exonuclease activity, by using phosphorothioation, and the like.

A method of using 5 '-3' exonuclease in the preparation of target nucleic acids is the subject of Nikiforov et al . , U.S. Patent 5,518,900, which is herein incorporated by reference. Examples of 5 '-3' exonuclease resistant nucleic acids are disclosed in Zon, G. et al . , (Anticancer Drug Design 6:539-568 (1991)) and Goodchild, J. et al. (Bioconjugate Chem. 1:613-629 (1990), both of which are incorporated herein by reference in their entirety. In general, suitable degradation-resistant nucleotide derivatives in which one or two of the non- bridging oxygen atoms of the phosphate moiety of a nucleotide have been replaced with a sulfur-containing group (especially a phosphorothioate) , an alkyl group (especially a methyl or ethyl alkyl group) , a nitrogen- containing group (especially an amine) , and/or a selenium-containing group, etc. Other modifications that confer degradation resistance such as 5 '-3' exonuclease resistance can be used, for example, peptide nucleic acids (PNAs) (Buchardt, 0. supra ) or 2 ' -O-methyl ribose modified nucleic acids Srivastava S., C. et al . , U.S. Pat. No. 5,214,135. Degradation resistance is preferably achieved by the primer containing a phosphorothioate modification at the 5' end of the nucleic acid and, in a preferred embodiment, containing four phosphorothioate linkages at the 5' end.

The selected nucleotide derivative is suitable for in vi tro primer-mediated extension and provides nuclease resistance to the region of the nucleic acid molecule in which it is incorporated. In a preferred embodiment, a nucleotide derivative confers resistance to exonucleases that attack double-stranded nucleic acids from the 5 ' -end (5'-3' exonucleases). Examples of such exonucleases include bacteriophage T7 gene 6 exonuclease and bacteriophage lambda exonuclease. Both exonucleases are inhibited by the presence of phosphorothioate bonds so as to allow the selected degradation of the unmodified nucleic acid strand. However, any double-strand specific, 5 '-3' exonuclease can be used for this process, provided its activity is affected by the presence of the modified nucleotides. The preferred enzyme when using phosphorothioate derivatives is the T7 gene 6 exonuclease, which shows maximal enzymatic activity in the same buffer used for many DNA dependent polymerase buffers including Taq polymerase. The 5 ' -3 ' exonuclease resistant properties of phosphorothioate derivative- containing DNA molecules are discussed, for example, in Kunkel, T.A. et al . (In: Nucleic Acids and Molecular Biology, Vol. 2, 124-135 (Eckstein F. et al . , eds . ) ,

Springer-Verlag, Berlin, (1988)). The 3 ' -5 ' exonuclease resistant properties of phosphorothioate nucleotide containing nucleic acid molecules are disclosed in Putney et al. (Proc. Natl. Acad. Sci. (U.S.A.) 78:7350-7354 (1981)) and Gupta A. P. et al. (Nucl. Acids Res., 12:5897- 5911 (1984) ) .

The amplification primers can be used to generate amplified nucleic acid products by any in vi tro amplification method known to those skilled in the art that uses a DNA dependent or RNA dependent DNA or RNA polymerase. The preferred method is the polymerase chain reaction (PCR) that involves template-dependent extension using thermally stable DNA polymerase (Mullis K et al, Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H et al.,EP 50,424; EP 84,796, EP 258,017, EP 237- 362; Mullis K., EP 201,184; Mullis, K. et al, U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al . , U.S. Pat. No. 4,683,194), incorporated herein by reference. PCR achieves the amplification of a specific nucleic acid sequence using two oligonucleotide primers complementary to regions of the sequence to be amplified. Extension products incorporating primers then become templates for subsequent amplification steps. Reviews of the polymerase chain reaction are provided by Mullis, K.B., ( supra ) ; Saiki, R.K. et al . , (Bio/Technology 3:1008-1012 (1985)); and Mullis, K.B. et al. (Meth. Enzymol. 155:335-350 (1987)), which is incorporated herein by reference.

Other nucleic acid amplification procedures can be used and include transcription-based amplification systems (Kwoh, D. et al . , Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989)); Gingeras T.R. et al., PCT appl . WO 88/10315 (priority: U.S. patent application Ser. Nos. 064,141 and 202,978); Miller, H.I. et al., PCT appl. WO 89/06700 (priority: U.S. patent application Ser. No. 146,462); Davey, C. et al . , (European Patent Application Publication No. 329,822)), RNA-dependent RNA amplification (Qβ replicase (Kramer, F.R. et al . , U.S. Pat. No. 4,786,600), and ligation-based amplification systems (Wu, D.Y. et al., Genomics 4:560 (1989)).

Amplification primers can be any length but are preferably as long as 90 nucleotides, preferably between 14 to 50 nucleotides, more preferably between 17 and 30 nucleotides.

In accordance with another embodiment of the invention, methods are provided for genotyping a nucleic acid sample comprising hybridizing an invention oligonucleotide to the nucleic acid sample, and preferably, performing a primer extension reaction .

As used herein, the term "genotyping" refers to determining the presence, absence or identity of a nucleotide base (e.g., a SNP) at a specific position in a target nucleic acid. First, a sample containing the target nucleic acid is treated, if such nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position. If the target nucleic acid is single-stranded, this step is not necessary. Second, the sample containing the target nucleic acid is contacted with a genotyping oligonucleotide (also referred to as a genotyping primer) under hybridizing conditions. The genotyping oligonucleotide is capable of hybridizing with a stretch of nucleotide bases present in the target nucleic acid, adjacent the nucleotide base to be identified (e.g., a SNP) , so as to form a duplex between the genotyping oligo and the target nucleic acid. When the genotyping oligonucleotide is "immediately adjacent" the nucleotide base to be identified (e.g., a SNP), the genotyping oligonucleotide hybridizes with the target nucleic acid in such a way that either the 3 ' or 5 ' end of the genotyping oligonucleotide is complementary to a nucleotide on the target nucleic acid that is located immediately 5' or 3', respectively, of the nucleotide base to be identified (see, e.g., Figure 2A and 2B) . It is also contemplated herein that the invention genotyping oligos can be fragments of the oligos in column 3 of the Table 1 (i.e., SEQ ID NO:4n+3, n=0-934) hybridizable to the target nucleic acid and adjacent to the nucleotide base to be identified such that the 3' end of the genotyping oligo is 1 up to 10, preferably 3 up to 6, nucleotides upstream from the nucleotide base to be identified in the target nucleic acid.

As used herein , the phrase "primer extension" refers to enzymatic extension of the genotyping primer in the resultant duplex by one or more nucleotides, catalyzed, for example, by a DNA polymerase, and the like. Such primer extension thus depends on correct base pairing of the added nucleotide to the nucleotide base to be identified. In one embodiment, the duplex of genotyping primer and target nucleic acid can then be contacted with a reagent containing at least two, but preferably four terminators, at least one of which terminators being labeled. The duplex of genotyping primer and the target nucleic acid is contacted with the reagent under conditions permitting base pairing of a complementary terminator present in the reagent with the nucleotide base to be identified and the occurrence of a template-dependent, primer extension reaction so as to incorporate a terminator at the 3' end of the primer. The net result is that the genotyping primer has been extended by one terminator. Next, the presence or absence of a labeled terminator at the 3 ' end of the extended genotyping primer is detected. The identity of the labeled terminator indicates which terminator has base paired to the next base in the target nucleic acid. Since the terminator is complementary to the next base in the target nucleic acid, the identity of the next base in the target nucleic acid is thereby determined.

It is also contemplated that the genotyping primer can be extended by a nucleic acid template- dependent ligation reaction. In such a reaction, a ligating oligonucleotide is used that hybridizes to the target nucleic acid only if the nucleotide residue corresponding to the SNP on the target molecule is complementary to the corresponding nucleotide on the ligating oligonucleotide. If such a ligating oligonucleotide hybridizes with the target nucleic acid, the ligating oligonucleotide and the genotyping oligonucleotide will be immediately adjacent each other. The genotyping primer and ligating primer can then be ligated by contacting the target nucleic acid, genotyping primer and ligating oligonucleotide with a nucleic acid ligase. The ligation reaction can ligate the 5' end of the genotyping primer to the 3' end of the ligating primer, or alternatively, the ligation reaction can ligate the 3' end of the genotyping primer to the 5' end of the ligating primer. Either the genotyping primer or the ligating oligonucleotide can be labeled with a detectable marker. The detection of a ligation product indicates the presence of the SNP nucleotide targeted by the ligation primer.

As used herein, a detectable marker is any molecule or structure that can be detected by spectroscopic, scattering, emission, absorption, binding, or other known detection methods known in the art. Exemplary detectable markers include a radionuclide, a fluorochrome, a colorimetric agent, a magnetic substance, an electron-rich material such as a metal, a luminescent tag, or a detectable binding agent such as biotin. Preferably, the detectable marker is a fluorochrome or a detectable binding agent. More preferably, the detectable marker is a fluorescent dye or a detectable binding agent that can be bound by an antibody. In one embodiment, the detectable marker is biotin or fluorescein. A detectable binding agent refers to any substance that can bind a solid support, a probe molecule, or other molecule that binds the detectable binding agent and, in turn, is then detected or permits identification of the nucleic acid which is labeled with the detectable binding agent. For example, a detectable marker can be biotin which is bound by an antibody specific to biotin, whereupon the antibody can then be detected in an ELISA-based assay. Other detectable markers known in the art may be used in the invention assay, for example, markers listed in the catalog of Molecular Probes (Eugene, OR) , markers listed in the catalog of Synthegen (Houston, TX) , and markers listed in WO 98/59066, which are all herein incorporated by reference .

It is further contemplated herein that one or more primer extension reactions, used in genotyping a nucleic acid sample comprising one or more different target nucleic acids, can be carried out in the same reaction vessel. In one embodiment, at least one terminator is labeled with a detectable marker. In another embodiment, one or more genotyping primers are each labeled with a detectable marker. Detection of identity of the SNPs on the one or more target nucleic acids can then be carried out using one of a variety of methods including spectroscopic detection of one or more detectable markers, separation of one or more nucleic acid sequences used to identify the identity of the corresponding one or more SNPs. Such methods for the detection of one or more SNPs in a single reaction vessel are described in WO 98/59066, which is herein incorporated by reference.

In a preferred embodiment, the primer extension reaction is a "single-nucleotide primer extension" as described, for example in United States Patent No. 5,679,524 and in United States Patent No. 5,888,819, incorporated herein by reference in their entirety. In this method, a genotyping primer hybridizes with the target nucleic acid immediately adjacent the nucleotide base to be identified, so as to form a duplex between the genotyping primer and the target nucleic acid such that the nucleotide base to be identified is the first unpaired base in the template target nucleic acid immediately downstream of the 3' end of the genotyping primer in the duplex of genotyping primer and target nucleic acid. In this embodiment, primer extension will add a single terminator residue which is complementary to the base to be determined in the target nucleic acid. Identification of the terminator will thus provide identification of the base on the target nucleic acid to be determined.

Typically, two or more terminators are labeled, each labeled with a different detectable marker, thereby permitting simultaneous detection (and hence, identification) of the two or more labeled terminators. Preferably four terminators are labeled, each with a different detectable marker, thereby permitting simultaneous detection and identification of up to four labeled terminators.

In the embodiment in which only one terminator is labeled (a first labeled terminator) , the above- described primer extension procedure can be repeated at least once, using a second labeled terminator that is different from the first labeled terminator. This process can be repeated once, twice or three times, using a different, labeled terminator for each repetition.

As used herein, the phrase "nucleic acid sample" refers to one or more of target nucleic acids, which can be from any source. The sample of target nucleic acids can be natural or synthetic (i.e., synthesized enzymatically in vitro) . The sample of target nucleic acids can comprise deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), or copolymers of deoxyribonucleic acid and ribonucleic acid. The target nucleic acid can be a deoxyribonucleic acid (DNA) , a ribonucleic acid (RNA) , or a copolymer of deoxyribonucleic acid and ribonucleic acid. The target nucleic acid can be synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically . The sample containing the target nucleic acid or acids can comprise genomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof. The sample containing the target nucleic acid or acids can also comprise extragenomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof. Preferably, the target nucleic acid or acids are synthesized by amplification methods described herein. More preferably, the target nucleic acid or acids are synthesized by the polymerase chain reaction.

The sample can be taken from any organism, but is preferably human. Some examples of organisms to which the method of the subject invention is applicable include plants, microorganisms, viruses, birds, vertebrates, invertebrates, mammals, humans, horses, dogs, cows, cats, pigs, or sheep. For assay of genomic DNA, virtually any biological tissue samples can be used, including whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. Genomic DNA is typically amplified by PCR, and the like, before analysis.

The target nucleic acid can comprise one or more moieties that permit affinity separation of the target nucleic acid from the unincorporated reagent and/or the primer. The target nucleic acid can comprise biotin which permits affinity separation of the target nucleic acid from the unincorporated reagent and/or the primer via binding of the biotin to streptavidin which is attached to a solid support. The sequence of the target nucleic acid can comprise a DNA sequence that permits affinity separation of the target nucleic acid from the unincorporated reagent and/or the primer via base pairing to a complementary sequence present in a nucleic acid attached to a solid support. The target nucleic acid can be labeled with a detectable marker; this detectable marker can be different from any detectable marker present in the reagent or attached to the primer.

In a preferred embodiment, the target nucleic acid sequence is synthesized by an amplification method and is modified at the 5' end to prevent hydrolysis by, for example, exonuclease activity, as described herein. See, for example, United States Patent 5,518,900, which describes a method for generating single-stranded nucleic acid molecules containing nuclease-resistant nucleotides.

The genotyping primer can comprise one or more moieties that permit affinity separation of the primer from the unincorporated reagent and/or the target nucleic acid. The genotyping primer can comprise biotin which permits affinity separation of the primer from the unincorporated reagent and/or target nucleic acid via binding of the biotin to streptavidin which is attached to a solid support. The sequence of the genotyping primer can comprise a DNA sequence that permits affinity separation of the primer from the unincorporated reagent and/or the target nucleic acid via base pairing to a complementary sequence present in a nucleic acid attached to a solid support.

In another embodiment of the invention, a method is provided for genotyping a nucleic acid sample comprising: a) amplifying a target nucleic acid sequence that hybridizes to an oligonucleotide selected from the group of SEQ ID NOs consisting of 4n+3, or complementary sequences thereof; and

b) performing a single-nucleotide primer extension reaction employing an oligonucleotide comprising a nucleic acid selected from the group of SEQ ID NOs consisting of 4n+3, or complementary sequences thereof,

wherein n=0 through 934 and is the same value in both steps a and b.

As used herein, a target nucleic acid sequence that hybridizes to an oligonucleotide selected from the group of SEQ ID NOs consisting of 4n+3 or complementary sequences thereof refers to a target nucleic acid comprising a sequence substantially similar to at least a portion of nucleic acid of SEQ ID NO:4n+4, or complementary sequences thereof that hybridizes to a nucleic acid of SEQ ID NO:4n+3. Typically, such a target nucleic acid sequence will comprise the nucleic acid of SEQ ID NO:4n+4 with the exception of the nucleotide immediately adjacent the 3' end of the nucleic acid of SEQ ID NO:4n+3, which may or may not be present, where n is the same value for both SEQ ID NOs:4n+3 and 4n+4.

In a further embodiment of the invention, a kit is provided which contains at least one genotyping primer. As used herein, a kit refers to a genotyping system in kit form, comprising at least one invention genotyping primer, and optionally further comprising chain termination reagent in combination with a DNA polymerase with or without an associated 3' to 5 ' exonuclease function, and an appropriate salt and cofactor mixture, in a suitable packaging material. Invention kits are useful for assaying for the presence or absence of a nucleotide at a specified location on one or more target nucleic acids . Preferably, the invention kit is useful for determining the identity of a nucleotide at a specified location on one or more target nucleic acids. To facilitate the assaying for the presence or absence, or determining the identity of the SNP nucleotide on the target nucleic acid, the invention kit provides a genotyping primer, one or more terminators, or both a genotyping primer and one or more terminators, that are labeled with a detectable marker.

The conditions for the occurrence of the template-dependent, primer extension reaction can be created, in part, by the presence of a suitable template-dependent enzyme. Some of the suitable template-dependent enzymes are DNA polymerases. The DNA polymerase can be of several types . The DNA polymerase must, however, be primer and template dependent. For example, E. coli DNA polymerase I or the "Klenow fragment" thereof, T4 DNA polymerase, T7 DNA polymerase ( "Sequenase" ) , Thermus aquaticus DNA polymerase, or a retroviral reverse transcriptase can be used. RNA polymerases such as T3 or T7 RNA polymerase can also be used in some protocols. Depending upon the polymerase, different solution conditions and different temperature ranges are used for the hybridization and extension reactions,

The reagents of the subject invention typically permit the typing of nucleic acids of interest by facilitating the analysis of the 3' terminal addition of terminators to a specific primer or primers under specific hybridization and primer extension conditions. Using only one terminator in the chain termination reagent as the nucleoside triphosphate substrate ensures addition of only one nucleotide residue to the 3' terminus of the primer in the polymerase reaction. Using all four terminators simultaneously ensures fidelity, i.e., suppression of misreading.

A genotyping primer of an invention kit comprises a genotyping primer as described above, which is capable of hybridizing to a target nucleic acid. An invention kit comprises one or more genotyping primers. In one embodiment of an invention kit, the genotyping primer (s) comprises any of SEQ ID NO:4n+3, or the complement thereof, where n=0 to 934. In another embodiment the genotyping primer (s) comprises a portion of any of SEQ ID NO:4n+4, or the complement thereof, where n=0 to 934. In yet another embodiment the genotyping primer (s) hybridizes with any of SEQ ID NO:4n+4, or the complement thereof, where n=0 to 934.

A suitable kit includes at least one invention genotyping primer, and optionally further comprising chain termination reagent in combination with a DNA polymerase with or without an associated 3' to 5 ' exonuclease function, and an appropriate salt and cofactor mixture, as a separately packaged chemical reagent (s) in an amount sufficient for at least one assay. Instructions for use of the packaged reagent are also typically included. Those of skill in the art can readily incorporate invention genotyping primers into kit form in combination with appropriate buffers and solutions for the practice of the invention methods as described herein. In another embodiment, an invention kit comprises a genotyping primer and two amplification primers. This kit can alsq comprise chain termination reagent in combination with a DNA polymerase with or without an associated 3' to 5 ' exonuclease function, and an appropriate salt and cofactor mixture to be used in conjunction with the genotyping primer, and can also comprise nucleotide reagent in combination with a DNA polymerase with or without an associated 3' to 5 ' exonuclease function, and an appropriate salt and cofactor mixture to be used in conjunction with the amplification primers, in a suitable packaging material. Such invention kits are useful for (1) amplifying one or more target nucleic acid sequences in a nucleic acid sample and (2) assaying for the presence or absence of a nucleotide at a specified location on one or more target nucleic acids. Preferably, invention kits are useful for (1) amplifying one or more target nucleic acid sequences in a nucleic acid sample and (2) determining the identity of a nucleotide at a specified location on one or more target nucleic acids.

Preferably, a kit comprising one genotyping primer and two amplification primers comprises a genotyping primer that hybridizes to the sequence SEQ ID NO:4n+4 and amplification primers of the sequence SEQ ID NOs:4n+l and 4n+2, where n=0 through 934 and the three oligonucleotides correspond to SEQ ID NOs having the same value for n.

More preferably, a kit comprising one genotyping primer and two amplification primers comprises a genotyping primer of the sequence SEQ ID NO:4n+3 and amplification primers of the sequence SEQ ID NOs:4n+l and 4n+2, where n=0 through 934 and the three oligonucleotides correspond to three consecutive SEQ ID NOs having the same value for n.

In yet another embodiment of the invention, a kit comprises two amplification primers. This kit can also comprise nucleotide reagent in combination with a DNA polymerase with or without an associated 3' to 5 ' exonuclease function, and an appropriate salt and cofactor mixture to be used in conjunction with the amplification primers, in a suitable packaging material. Such invention kits are useful for amplifying one or more target nucleic acid sequences in a nucleic acid sample.

A termination reagent of an invention kit refers to a reagent comprising at least one terminator. Preferably, a termination reagent comprises two terminators. More preferably, a termination reagent comprises three terminators. Most preferably, a termination reagent comprises four terminators. A termination reagent will comprise one or more terminators labeled with a detectable marker. Preferably, a termination reagent comprises two or more terminators labeled with a detectable marker. More preferably, a termination reagent comprises three or more terminators labeled with a detectable marker. Most preferably, a termination reagent comprises four or more terminators labeled with a detectable marker.

A nucleotide reagent of an invention kit for use in an amplification reaction refers to a reagent comprising at least one nucleotide triphosphate . Preferably, a nucleotide reagent comprises two nucleotide triphosphates . More preferably, a nucleotide reagent comprises three nucleotide triphosphates. Most preferably, a nucleotide reagent comprises four nucleotide triphosphates .

In a particularly preferred embodiment, an invention kit comprises a collection reagents termed "Master Mix" as described in Example I. Exemplary Master Mix 2x is as follows: 768 mL of sterile, distilled water 200 mL of lOxPCR salts (100 mM Tris-HCl, pH 8.5, 500 mM KC1, 15 mM MgCl₂ and 0.01% Gelatin) and 8 mL of each dNTP stock (100 mM stocks of deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP) in sterile, distilled H₂0.

The final composition being: 50 mM Tris-HCl, pH 8.5, 100 mM KC1, 3 mM MgCl₂, 0.002% Gelatin, and 800 μM each dNTP. This solution can be stored at -20°C until use. Master mix can be prepared in a variety of different concentrations, described as a function of x, as the above example depicts 2x Master Mix. The concentration of the master mix employed herein may vary from 0.5x, lx, 2x, or 5x, to as high as lOx, 20x, or 50x. Preferably, the master mix is from 2x to 5x.

In addition, in place of Tris-HCl, any other buffer which does not interfere with the enzymatic and/or detection processes of required to detect the presence, absence or identity of a SNP may be used. Such buffers include the so-called "Good buffers", and other organo- amino based buffers, and the like. Similarly, the pH may be adjusted to any range capable of carrying out the desired processes. The pH may range from as low as about 6.0, 7.0, or 8.0, to as high as about 8.5, 8.7, or 9.0. Preferably, the pH ranges from about 7.0 to about 8.7. More preferably, the pH ranges from about 8.0 to about

8.5. Buffer concentration may vary from as low as 10 mM, 25 mM or 50 mM, to as high as 75 mM, 100 mM or 200 mM. Preferably, the buffer concentration is from 25 mM to 100 mM.

In place of KC1, other monovalent-cation salts can be used. Other exemplary monovalent-cation salts include salts with the cation: NH₄ ⁺, alkalai metal ions Li⁺, Na⁺, Rb⁺, Cs⁺, and other monovalent cations. The anion can be any anion which does not adversely influence the amplification reaction. Exemplary anions are halide anions, F^", Cl^", Br^", I^", and other anions such as phosphate, sulfate, nitrate, and the like. The concentration of the monovalent salt can be as low as 15 mM, 40 mM, or 75 mM, to as high as 100 mM, 150 mM or 250 mM. Preferably, the monovalent-cation salt concentration is from about 75 to 150 mM.

In place of MgCl₂, other divalent-cation salts can be used. Other exemplary divalent-cation salts include salts with the cation: Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and other divalent cations. The anion can be any anion which does not adversely influence the amplification reaction. Exemplary anions are halide anions, F^", Cl^", Br^", I^", and other anions such as phosphate, sulfate, nitrate, and the like. The concentration of the divalent salt can be as low as 0.5 mM, 1 mM, or 3 mM, to as high as 5 mM, 10 mM or 25 mM. Preferably, the divalent-cation salt concentration is from about 1 mM to 10 mM.

In place of gelatin, other volume exclusion agents can be used such as polyvinyl pyrrolidone, polyethlene glycol, polyacrylamide, linear polyacrylamide, and the like. The volume exclusion reagent can be absent, or can be as low as 0.0001%, 0.0005% or 0.001%, to as high as 0.002%, 0.005% or 0.02%. Preferably, the volume exclusion agent is about 0.001% to 0.005%.

The concentration of each of dCTP, dGTP, dATP and dTTP (i.e., the dNTPs) can vary in concentration from 250 μM, 500 μM, or 800 μM, to 1 mM, 1.5 mM or 2.5 mM. Preferably, the concentration of the dNTPs is about 500 μM to 1 mM.

As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as invention primers, and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the invention probes can be used for determining the presence or absence of, or preferably the identity of, a particular nucleotide on a target nucleic acid, thereby genotyping the target nucleic acid. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to determine the presence or absence of, or preferably the identity of, a particular nucleotide on a target nucleic acid.

The packaging materials employed herein in relation to diagnostic systems are those customarily utilized in nucleic acid-based assay systems. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits an isolated nucleic acid, oligonucleotide, or primer of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated nucleic acid, oligonucleotide or primer, or it can be a microtiter plate well to which microgram quantities of a contemplated nucleic acid probe have been operatively affixed.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.

It is contemplated herein that each of the genotyping and amplification primers described herein can be used in the invention methods of genotyping nucleic acid samples, for example, to assess by association analysis the genotype of an individual, or group of individuals, having a pathological phenotypic trait suspected of being caused by one or more single nucleotide polymorphisms. Phenotypic traits suitable for association analysis include diseases that have known but yet unmapped genetic components, e. g., agammaglobulinemia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent porphyria, and the like.

Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, system, diseases of the nervous system and infection by pathogenic microorganisms. Some examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulindependent and non-independent), systemic lupus erythematosus and Graves disease, and the like. Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, oral cavity, ovary, pancreas, prostate, skin, stomach, leukemia, liver, lung, and uterus, and the like.

Phenotypic traits also include characteristics such as longevity, appearance (e. g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments .

Such correlations can be exploited in several ways. In the case of a strong correlation between a polymorphic form and a disease for which treatment is available, detection of the polymorphic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring of the patient. Detection of a polymorphic form correlated with serious disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions. For example, the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymorphism from her husband to her offspring. In the case of a weaker, but still statistically significant correlation between a polymorphic set and human disease, immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the patient can be motivated to begin simple life-style changes (e. g., diet, exercise) that can be accomplished at little cost to the patient but confer potential benefits in reducing the risk of conditions to which the patient may have increased susceptibility by virtue of variant alleles. After determining polymorphic form(s) present in an individual at one or more polymorphic sites, this information can be used in a number of methods .

In addition, determination of which polymorphic forms occupy a set of polymorphic sites in an individual identifies a set of polymorphic forms that distinguishes the individual. See generally, National Research Council, The Evaluation of Forensic DNA Evidence (Eds.

Pollard et al . , National Academy Press, DC, 1996). Thus, each of the genotyping and amplification primers described herein can be used in the invention methods of genotyping nucleic acid samples, for example, to identify a distinguishing or unique set of forensic markers in an individual useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymorphic forms occupying selected polymorphic sites is the same in the suspect and the sample. If the set of polymorphic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source of the sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene. If frequencies of the polymorphic forms at the loci tested have been determined (e. g., by analysis of a suitable population of individuals) , one can perform a statistical analysis to determine the probability that a match of suspect and crime scene sample would occur by chance (see, e.g, WO 95/12607). If several polymorphic loci are tested, the cumulative probability of non- identity for random individuals becomes very high (e. g., one billion to one) . Such probabilities can be taken into account together with other evidence in determining the guilt or innocence of the suspect.

In addition, each of the genotyping and amplification primers described herein can be used in the invention methods of genotyping nucleic acid samples, for example, to identify a distinguishing or unique set of markers in an individual useful for paternity analysis. The object of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part of the child's genotype not attributable to the mother is consistent with that of the putative father. Paternity testing can be performed by analyzing sets of polymorphisms in the putative father and the child. If the set of polymorphisms in the child attributable to the father does not match the putative father, it can be concluded, barring experimental error, that the putative father is not the real father. If the set of polymorphisms in the child attributable to the father does match the set of polymorphisms of the putative father, a statistical calculation can be performed to determine the probability of coincidental match (see, e.g., WO 95/12607) . If several polymorphic loci are included in the analysis, the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of a putative father whose polymorphic marker set matches the child's polymorphic marker set attributable to his/her father. The single nucleotide polymorphisms set forth in column 4 of Table 1 (also referred to herein as SEQ ID NO:4n+4, wherein n=0 through 934) may contribute to the phenotype of an organism in different ways. Some of these polymorphisms may occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Others of these polymorphisms may occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymorphism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymorphisms in different genes. Further, some of these polymorphisms may predispose an individual to a distinct mutation that is causally related to a certain phenotype.

Phenotypic traits include diseases that have known but yet unmapped genetic components. Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms. Some examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non- independent) , systemic lupus erythematosus and Graves disease. Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, stomach and uterus. Phenotypic traits also include characteristics such as longevity, appearance (e. g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments .

Correlation is performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for polymorphic markers sets. To perform such analysis, the presence or absence of a single or a set of polymorphisms (i. e. a polymorphic set) is determined for a set of the individuals, some of whom exhibit a particular trait, and some of which exhibit lack of the trait. The alleles of each polymorphism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest.

Correlation can be performed by standard statistical methods such as a chi-squared test and statistically significant correlations between polymorphic form(s) and phenotypic characteristics are noted. For example, it might be found that the presence of allele Al at polymorphism A correlates with heart disease. Such correlations can be exploited in several ways as set forth above.

For example, in the case of a strong correlation between a set of one or more polymorphic forms and a disease for which treatment is available, detection of the polymorphic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring of the patient. Thus, the methods of genotyping nucleic acid samples described herein can be used, for example, to diagnose any of more than 3000 genetic diseases currently known or to be identified, e.g., hemophilias, thalassemias, Duchene Muscular Dystrophy (DMD) , Huntington' s Disease (HD) , Alzheimer's Disease and Cystic Fibrosis (CF) , and the like.

It is also contemplated herein that each of the genotyping and amplification primers described herein can be used in the invention methods of genotyping nucleic acid samples, for example, for assessing the pharmacogenomic susceptibility of a subject harboring a single nucleotide polymorphism to a particular pharmaceutical compound, or to a class of such compounds. Genetic polymorphism in drug metabolizing enzymes, drug transporters, receptors for pharmaceutical agents, and other drug targets have been correlated with individual differences based on distinction in the efficacy and toxicity of the pharmaceutical agent administered to a subject. Pharmocogenomic characterization of a subjects susceptibility to a drug enhances the ability to tailor a dosing regimen to the particular genetic constitution of the subject, thereby enhancing and optimizing the therapeutic effectiveness of the therapy.

All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto. The invention will now be described in greater detail by reference to the following non-limiting examples. Examples

Example I

Preparation of 2x Master Mix for PCR

A component of genotyping using SNPE is accurate and efficient amplification of the nucleic acid region being tested for the SNP. To facilitate control of the PCR used, a reaction mixture, referred to herein as "2x Master Mix," has been produced to achieve more successful PCRs .

2x Master Mix was prepared as follows: lOxPCR salts was 100 mM Tris-HCl, pH 8.5, 500 mM KCl, 15 mM MgCl₂ and 0.01% Gelatin, stored in a -20°C freezer. 100 mM stocks of deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP (dNTPs) (LTI, Bethesda, MD) were made in sterile, distilled H₂0. To 768 mL of sterile, distilled water 200 mL of lOxPCR salts and 8 mL of each dNTP stock was added. The final composition was: 50 mM Tris-HCl, pH 8.5, 100 mM KCl, 3 mM MgCl₂, 0.002% Gelatin, and 800 μM each dNTP. The solution was stored at -20°C until use.

Example II

Single Nucleotide Polymorphism Detection by Single Nucleotide Primer Extension (SNPE)

Single nucleotide polymorphisms are the most common form of genetic diversity and are thought to be direct and indirect markers of many human diseases. The following example shows the use of Single Nucleotide Primer Extension (SNPE) to identify the presence of SNPs. The method of SNPE is the subject of US Patent 5,888,819 and more recently described in detail by Reynolds et al . in DNA Markers : Protocol s , Appl ica tions , and Overviews (ed. G. Caetano-Anolles) , pp. 199-211, Wiley-Liss, New York, NY (1997), both of which are herein incorporated by reference .

The amplification primers listed herein (Table 1, columns 1 and 2) were selected to amplify the corresponding SNP-containing oligo in column 4, forming a PCR amplified target nucleic acid such that the primers do not overlap the corresponding genotyping (SNPE) primer in column 3.

SNPE primers were designed to be complementary to the phosphorothioate-modified strand of the PCR product target nucleic acid and ending one nucleotide base short 3' to the polymorphic site to be interrogated as described (Reynolds et al . , supra ) . Either DNA strand can be a target for the SNPE primer as long as the phosphorothioate-modified strand of the PCR product is the complementary strand.

All oligonucleotides were synthesized by standard phosphoramidite chemistry on a PE Biosystems 392/394 DNA Synthesizer using Glen Research (Sterling, VA) reagents. Tetraethylthiuram sulfide (TETD; PE Biosystems, Foster City, CA) was used for phosphorothioate modification as per manufacturer' s instructions. All oligonucleotides were deprotected in concentrated ammonia and desalted using NAP5 or NAP25 gel filtration columns (Amersham Pharmacia Biotech,

Piscataway, NJ) as per manufacturer's instructions. Genomic DNA from genetically diverse human individuals purchased from the Coriell Institute for Medical Research (Camden, NJ) was the source of DNA for PCR amplifications. The DNA sample was diluted with sterile, distilled H₂0 to a final concentration of 5 ng/μL before use.

PCR amplification of 10 ng of template genomic DNA was performed in 384-well plates under the following conditions: 10 mM Tris-HCl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 400 μM each of dATP, dCTP, dGTP, and dTTP, (from the 2x Master Mix) and 0.050 U/μL Platinum Taq { Thermus aqua ticus) DNA polymerase, (LTI, Bethesda, MD) using 0.5 μM each primer concentration in a total volume of 5 μL. The PCR amplification was performed in a PTC-225 DNA Engine Tetrad thermocycler (MJ Research, Watertown, MA) using the following protocol: following a 2 minute denaturation step at 95°C thirty- five cycles were carried out, each consisting of denaturation (30 seconds at 94°C), annealing (2 minutes at 55°C) , and extension (30 seconds at 72°C) steps. This was followed by a final extension step (1 min at 72°C) and hold (4°C) .

Single stranded target nucleic acid DNA was prepared from the double-stranded PCR product by treatment with a 5 '-3' DNA exonuclease. Briefly, 5 μL T7 gene 6 exonuclease (United States Biologicals, Swampscott, MA) in 0.5 M Tris-HCl, pH 7.5, 1 mM Dithiothreitol (DTT), and 0.01% acetylated Bovine Serum Albumin (BSA) was added to a final concentration of 0.45 U/μL and incubated for one hour at room temperature.

The reaction was terminated by the addition of 5 μL of 3 x SNPE Salt (4.5M NaCl, 30 mM EDTA, and 3 mM cetyltrimethylammonium bromide (CTAB) ) . The DNA created by PCR originating from the phosphorothioated primer is protected from nuclease digestion whereas the opposite strand with the unmodified primer is digested and hence will not interfere with the hybridizing strand to the immobilized genotyping (SNPE) primer.

The SNPE primer of interest was attached to a polystyrene 384-well plate (NUNC) . Briefly, 20 μL aliquots of 0.25 μM SNPE oligonucleotide in 50 mM N,N- dimethyloctylamine hydrochloride, pH 7.0 (ODA; Segma- Aldrich, Milwaukee, WI ) was added to each well and incubated overnight at 37°C. The plates were washed with TNTw (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween- 20) three times and once with lxTE/ISOH (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% isopropanol ) . The plates were incubated upside down at 50°C for 30 minutes prior to storage in sealed pouch with dessicant. 15 μL of the exonuclease digestion product was added to the appropriate well and the single-stranded PCR product was allowed to hybridize for 30 minutes at room temperature. All wells were subsequently washed with TNTw three times.

An exonuclease (-) version of the Klenow Fragment of E. coli DNA Polymerase I (United States Biologicals, Swampscott, MA) was used for template- directed single nucleotide extension of the SNPE primer. Briefly, the enzyme was diluted in a buffer containing 10 mM Tris-HCl, pH 7.5, 5 mM DTT and 0.5 mg/mL BSA. The extension reaction was performed in 20mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 25mM NaCl, 10 mM MnCl₂, 15 mM Sodium Isocitrate, 1.5 μM each of the four 2', 3'- dideoxynucleoside 5 ' -triphosphates (ddNTPs), ddATP, ddCTP, ddTTP, ddGTP. Two of the ddNTPs were unlabeled, one was labeled with biotin, e.g. 2', 3 ' -dideoxyuridine 5 ' -triphosphate (biotin-ddUTP) , and one labeled with fluorescein, e.g. fluorescein-labeled 2', 3'- dideoxycytosine 5 ' -triphosphate (fluoresceinated-ddCTP) . The determination of which labeled ddNTPs to use depends on the nucleotide identity of the presumed SNP. For example, a suspected C to T mutation can be assayed using a mix of ddATP, fluorescein-ddCTP, ddGTP, and biotin- ddUTP. The enzyme concentration was 0.02 U/μL and the reaction was performed at room temperature for 30 minutes. The plates were washed with TNTw three times, then 0.2N NaOH, and then three times with TNTw.

Detection of the extended primers was performed by standard ELISA techniques using two nucleotide- specific labeled antibody conjugates. The dilutions of each are determined empirically for each antibody lot to achieve approximately equal intensity for the two colorimetric signals in the presence of both alleles. Briefly, the wells were incubated for 30 minutes at room temperature with 10 μL of 1% fraction V BSA (Sigma, St. Louis, MO) in TNTw containing an alkaline phosphatase conjugate of antifluorescein (1:2000 dilution)

(Boehringer Mannheim, Indianapolis, IN) . After washing with TNTw six times, the presence of alkaline phosphatase was determined first by the addition of 25 μL per well of a 1.5 mg/mL solution of p-nitrophenyl phosphate (Moss, Pasadena, MD) in 100 mM diethanolamine, pH 9.5, 20 mM

MgCl₂. The plate was immediately placed in a microplate reader (ICN, Costa Mesa, CA) and the development of color monitored spectrophotometrically at a wavelength of 405 nm after 24 minutes in an endpoint assay. Next, the detection of biotinylated-ddNTP was performed. The wells were incubated for 30 minutes at room temperature with 10 μL of 1% fraction V BSA in TNTw containing a horseradish peroxidase-conjugated antibiotin (1:500 dilution) (Zymed, San Francisco, CA) . The plates were then washed three times with TNTw and the presence of fluorescein was determined by the addition of 25 μL of a 1 mg/ml solution of tetramethylbenzidine (TMB; Moss, Pasadena, MD) . The plate was immediately placed in a microplate reader and the development of color monitored spectrophotometrically at a wavelength of 620 nm after 24 minutes in an endpoint assay.

A scatterplot of the raw data of OD₄₀₅ v. OD₆₂₀ was used in the visual determination of sample genotypes. A polymorphism is identified when three distinct clusters are seen in the positive regions of the scatterplot. These correspond to individuals that are homozygous, forming one cluster that is positive for OD₄₀₅ and another that is positive for OD₆₂₀, or heterozygous, forming a third cluster that is positive for OD₄₀₅ and OD₅₂₀.

Figure 3 shows a scatterplot that was classified polymorphic. The PCR primers used were oligonucleotides of SEQ ID NOS:25 and 26 (Ref No. 261, Table 1) . The SNPE primer used was an oligonucleotide with SEQ ID NO:27. The targeted nucleic acid with the suspected polymorphism contains the sequence of SEQ ID NO: 28. SNPE was performed using fluorescein-ddCTP and biotin-ddUTP . The Y-axis corresponds to signal obtained from ddUTP incorporation and the X-axis corresponds to signal obtained from ddCTP incorporation. Note that there are clusters where each allele tested is positive (CC or TT) and where both alleles are positive (CT) . This is an example where a SNP is identified. Note that there is another cluster about the origin which corresponds to no signal (NS) due to PCR failure.

Table 1 lists the invention oligonucleotides disclosed herein. Each row provides two PCR primers used to amplify the target DNA comprising a certain SNP (Columns 1 and 2), one genotyping oligonucleotide (Column 3; referred to as a SNPE primer) used to probe the identity of the base at the location in the sequence corresponding to that SNP) , and sequence of the region flanking the site of that SNP (Column 4) . A "P" listed to the right of a PCR primer denotes a phosphorothioation of that primer at its 5' end. In this embodiment, such a phosphorothioation refers to the five most 5' nucleotides of one of the two PCR primers being linked by four exonuclease-resistant phosphorothioated linkages (e.g., 5' C- (p) -G- (p) -C- (p) -A- (p) -GTCTCAGGCCAGCT 3', for SEQ ID NO:2, where "- (p) -" represents a phosphorothioate linkage) . While phosphorothioation sites are listed for the specific residues herein, it is understood that other modifications which confer exonuclease resistance can serve as equally desirable substitutes.

In this particular embodiment, nucleotides labeled as "X" refer to C3 linkers placed in the corresponding sequences at the location of the X. While C3 linkers are listed herein, it is understood that other single-nucleotide-spacers can serve as equally desirable substitutes .

The sequences of Table 1 are arranged in the same manner as the SEQ ID NOs are listed. Accordingly, the nucleic acids of SEQ ID NOs:l, 2, 3 and 4 correspond to the nucleic acids in Row 1, Columns 1, 2, 3 and 4, respectively. Similarly, the nucleic acids of SEQ ID NOs: 5, 6, 7 and 8 correspond to the nucleic acids in Row 2, Columns 1, 2, 3 and 4, respectively. Therefore, a general formula can be established to describe the SEQ ID NO of each nucleic acid:

SEQ ID NO= (4 x (Row Number - 1) ) + Column Number

RθW# REF# Upper PCR primer Modified Lower PCR primer Modified primer Flanking sequence

1 255 TTGAAATGTGAATAGTAAACTATGTAGAGATGA GGCAGTCTCAGGCCAGCT CAGTATGGGAAAAAAGCAXCTCGGGAG GTATGGGAAAAAAGCAGCTCGGGAGCTAGGGTGCTGAGCAGAGAAGAGTGA

2 256 CTCTTGGCTAGTTTATGTGAGATGAGA AGCTTTCAATCCTTCCAAACTTG TCCTACTTCATCACTTCTTCTATTA TCCTACTTCATCACTTCTTCTATTACGACTAATGTCTTCTGATGTTTACCA

3 257 CTACTGACTGCTTGACTGAGCTAATTAAT AATACATCAGAACCCCAAATCACA GCTTGXCCTCTTGTCATAATCAGCC GCTTGGCCTCTTGTCATAATCAGCCCGAGCGGGGATAGCTCACAAGACCAT

4 258 AGTTATTGCTTTCGTGAGGTGAAG TCAAAGGAAAAGGTATGTGGAATATT AAGTGCATCGGCCTTCAGAACGAGO ATTTTGCTGTAGTAATCTGTTGCACACCTCGTTCTGAAGGCCGATGCACTT

5 259 ATCTCAATTCCACGTTTOCTCT CATGGAGGGAAAATCCTTAACTTT CTTTGCGAACAGAGGTCCCTGGAGG AGGGGGAGCCTCAGCTCCACTGTCTCCCTCCAGGGACCTCTGTTCGCAAAG

6 260 AAGCAGCTCTGACTTTTGCAGT CCCCTGGACCATAACTTCAGT GTGTTXCACATAAAGTCAGCACATTTT CTGACTTCAAGACGTCATTGATCTTAAAAATGTGCTGACTTTATGTGCAAC

7 261 GTGGATATAGTAATTATCAGACATGGACATTA ATTTTATTGTTTTGTGGCTCTCAGTT ATTTATTCAAGAAAXCAGATGATAAGA TTATTCAAGAAATCAGATGATAAGACAGAGGACTTCAGCATAGAACTGAGA

8 262 CGAGCATCAGGTTATTGAAGC CTAGGGAGTTAGCCCCGCT CAGAGAGGCCTGTGAAGAGCTACCC CAGAGAGGCCTGTGAAGAGCTACCCGTGGATGCAGTGAGTGTGGGGGTAGA

9 263 CAAGTGAGTAAGGGGGCTTG CTCACATCAGCCTTCTCCATC TTAGAACACTGGGGGATTTACCTTG TTAGAACACTGGGGGATTTACCTTGCACTTTTTAAAAATAAGACTCATGAT

10 265 TGAAGGAAGGCGGCTGCA GAAACTCTCTTTCCCACAAAGGA CAXAGXAGGGTCTCCTTTTTCTACTCC CAAATACTTTGAGTGCTGCATCTTTGGGAGTAGAAAAAGGAGACCCTGCTT

11 266 GTGGTGGAGTCAGGACATGAGTA ACACCAAAGGGTTTTTGTGATAATC AATGAGATAAGGGCTATXAAAAGTTTA AATAACTTATTTTGCAAATATTGTACTAAACTTTTAATAGCCCTTATCTCA

12 267 TATTTGCCAAACCTTTCGC GAAAAAAACCAAAGGGGTGC AATACCACACTATTTGATTCCTGTA AATACCACACTATTTGATTCCTGTAGCTTCAGAGTAAGTCTTGAAGTCAGG

13 269 AGTGAAAAGGATCRGTCACAGC CATATAAATCAGGAGCAGAAWCAGAGA CAGAGAAGGCCCCCAAACTCAGACA AAAATGTTTCTGATGTGAATTTAAAGTGTCTGAGTTTGGGGGCCTTCTCTG

14 270 AGGATCRGTCACAGCAATAACTTAAG AAACTCAGACAYTTTAAATTCACATCAGA TTCACATCAGAAACATTTTGCCACT AATGGACATATGTATCCTAAGAAGACAGTGGCAAAATGTTTCTGATGTGAA

15 271 ATGGTTTAAAAGTGTGTGGGACTTC CATGGCAGAAGGTGAAGGG ACTTTTGCTCACTCTCTCTCTCCTG ACTTTTGCTCACTCTCTCTCTCCTGCCACCATGTGAATAAAGTGTTTGCTT

16 272 TGACTGGCAGATTGACAAAGAA CTATGGCGCTGCATGGCA AGTCXTCTAAATGCATGAGGGTGAAGC TCTTCTAAATGCATGAGGGTGAAGCGTGGGAACTGCTGTAACAGGCCCCAT

17 273 TGTACCTGCTTGTCATATGCTGAT GTTACTGATGGAGGGGTGGATATAC ATAGATGGATGGATGGATAGATGGA CATTCACCTGTCTTTCTATCCTTCCGTCCATCTATCCATCCATCCATCTAT

18 274 GAACTTGATTTCCTATGTGAACCTTATATT TCTATTACTATTTATGCACTGGAATACTTCTATAA TTCATTTAGAGXAATTTTACAGTTGAT CATTTAGAGTAATTTTACAGTTGATGGGGGAATATATTTTTTATAGAAGTA

19 275 AAATTTGGGTGGGGACACA CTAAGATATGGAGCAGGTACCCC ATGCAAACCCTCCACAACAACAAAA ACCGTATCACTTAACAATGTATGCAGTTTTGTTGTTGTGGAGGGTTTGCAT

20 276 GTGGAACAGAATAGAGAGACCAGAAA TGCAAGTGTGTGTCCAATAGTTCT TTGTTCCATTGTATTGTGCCTTTTC AATGTAAATATAACCAAGTGATCTTCGAAAAGGCACAATACAATGGAACAA

21 277 ATATAATCATTCAGCAGATATTTATCGATTG GCTGGATCAGGGCTCCTT TTGGGTGCCCACTAGCCTTGCCTTT TAATAAGAATAAAGGAGGCTGTGATCAAAGGCAAGGCTAGTGGGCACCCAA

22 278 TCCTATGATCTTAAACTTCCCTGTACC CAATGAAATGGGTATGAAAGTGCT GTGCTGTATGATGXCACTTCTGGGCGT CTCTGGATTTGAGGTTTGCATGGGTCACGCCCAGAAGTGGCATCATACAGC

23 279 GGGTTTACATTTCTGCTTAATCAATAT TGGGAATAGATACGCCCACA ATTCAGAATTTACTXCTACAGCACAAT CAGCATATGTTGTTTAAATATAATTGATTGTGCTGTAGCAGTAAATTCTGA

24 280 GATCCTACTTTGGGACCAGGT CCAGGAGTGGAAGAGATCGC GAATTTTTTXATTGATTATACACTATA TCTATTGTACCCTCCCATGAAGCCAGTATAGTGTATAATCAATTAAAAAAT

25 281 CTAGTTCTTTTGGAAAAGATAAGCCAA TCTTCACCCTAGTTTGCACAATTC CATGACTCTTAAGTGGATGAAAGTT CATGACTCTTAAGTGGATGAAAGTTGAGTGTGCAGATGTCTGCATGAATTG

26 282 ATTAAGTGCCTCACGTAATACTGGG CTTCGGCCTTCTCAGGGC TGGGACGCTGGAGAGACACAAGGAA TGGGACGCTGGAGAGACACAAGGAAACCCAAGATTATTTACTAGCCTTAAT

27 283 TGGGGTTACAGAAACTTACCATTACA ACCACTGCACTCCAGCCTG AAGTGTGACTCCGTCTCAAAAAAAA AAAGATGTTTTATTGTTTTTTTTTGGTTTTTTTTGAGACGGAGTCACACTT

28 284 TGTGACTGGCTAAATCACTTTGAG CCAGATGGATTTTCTGCACC CACXAGGACTTGAAGTCTTGGAACATC TGCAAGACTAGAGTGCTTCCAGCACAGATGTTCCAAGACTTCAAGTCCTGG

29 285 AGCAACCAAAAAAAAAAATGTGTAGA CTAAGAATGGAAATCCTGCCCT AGGAAAAGCAAACGGATATTGTCTG AGGAAAAGCAAACGGATATTGTCTGCGTGTAGAGCCGTGGGGCCACTAGGG

30 286 CTGGGGCAGAGGCTAAACA CAAAATACACATACCTTTCCCAGTAAC TTGTTGCTTCAAGCCAAGAACCTTA TTGTTGCTTCAAGCCAAGAACCTTACGGCTGGGTGAAAATTCAAGACTCTG

31 ₂87 CCATTTCATGTGCTAAAAACCAG TTTTGGAATTTTTCATAGTGTTCCC TGTTCCCAAACTTGAYACTCATAGG ATCAAAGTAAAACCTGTTACAAATAGCCTATGAGTRTCAAGTTTGGGAACA

32 288 TCAGCCATGATTGTGAGGC TGCTATAAAGACATACCCAAGACTGG ATGTGGAACTGTGAGTCAXTTAAACCT GTGGAACTGTGAGTCAATTAAACCTCTTTTCTTTATAAATTACCCAGTCTT

33 289 CTCACCACCTACAACTTGRTATTTAGC GTATTTCTCATCTAAATGAAAGCACATTTT AAAGCACATTTTTAAGAGAGAAAGA ACTTTAATATTTCTATATCTTACACGTCTTTCTCTCTTAAAAATGTGCTTT

34 290 CAGAAAAATAGAAGCAATAGGACAGAC ATATGGTGGGTGGGACTTAGC CTATAGATACAGATGATATTTATTA CTATAGATACAGATGATATTTATTACGGGAATTGGCTCATGTGATTATGGA

35 291 ACCTCATGCATTACAAGGCTCTT GAAAGTTGGCAGGTACAAGGTCT TCTGGTTGCACCXCCATXCCCTGGGCA TGGTTGCACCCCCATOCCOTGGGCACGTGAGAAATTCATGAGCCCCATTCT

36 292 GCTACCATCTTCTTTGGCTATCATT TCAATGTTATCGATCAATGTGTCATAG AATGCXCAAATGGTATGAGTGTGTGTT GAAAATGCTCTATTCATTAAAACACAAACACACACTCATACCATTTGAGCA

37 293 GGGCTTCAGCAAAATACAGGT CCGGGGATCTGAGTGACA TGGTAAAAAAXGATTTCATTGTGATTA TGAAAGAGACATCAAAAATTTTAACGTAATCACAATGAAATCATTTTTTAC

38 294 ATTCAGTCCAGGGCTGGG GCCATCTAGCATGAACACATAGC ATAGCCATGAGCCATCAATGAGTCC GTCACTGGGAAAGACAACCTCCAGCCGGACTCATTGATGGCTCATGGCTAT

39 295 CTTCAAGCCTCCACTACATCCTC AGGTAGGAGAAACATGAGAGTTTTTTTAA TCCAATTTCTTGATAAACATGCAAC TCCAATTTCTTGATAAACATGCAACGTATTTTTTTCTGAGAAAATAGAAGC

40 296 CATGGGGAGAGAGAATCTAGTAAGTG TCCAGAGGCAGTTCAGAATAAAAA AAAAAAATXATGTGTTGATTAGAGACT AACAAATATGTGTCTGTAGCTGCAGGAGTCTCTAATCAACACATTATTTTT

41 297 GCATCTTGTTTTGAGATTCCATG AGAGGGAATGATCAGTCAGGG GGGAAATTCATCTXTAGACATTCATTA GGAAAGTGACAAATTGCAAGGAAACGTAATGAATGTCTATAGATGAATTTC

42 298 CTCACAGCACCTCAGCACC GTATACCAGGCAAGGTAGAGAAAGG AAACCCCCTCTATCCTCAGTGTGTC AAACCCCCTCTATCCTCAGTGTGTCCCCCAAAATAAGAAAAAATGTTTTAT

43 299 AGCACCTAAACCCCCTCTATCC CTGGGAATGGAAGAAAGCAGA CAGACTTTTAAGXATGTTTGCCTCATA GAAAAAATGTTTTATAATGAGCACACTATGAGGCAAACATGCTTAAAAGTC

44 300 TACAGGGGATTTTGGATCTTTGT GTTTAATCCTCAGAACAATCTCTCCTACT GGCAGAAGTGGCCCTATXAAGACACAT AGCTTATCTCCCACTTTCTGGCATGCATGTGTCTTAATAGGGCCACTTCTG

45 301 TTGATCTTACTCAGCTCTCTTCAGAG ACCCTGATTACAAGGGTTATAAAGAAG AGATCCAGCTTAGATTCCCATTCCA AGATCCAGCTTAGATTCCCATTCCACCTGCTGTCTCAGTATTCTTCTTTAT

46 302 TTAAAATGCAGACACTTCTTAATATATCGG TTTTAAGATTTTCAGAGGCAGGTTC CAGGTTCTGAATAGCCTTTGGTCTA TCTCAGTCATGAGGCTAAATTAGCCGTAGACCAAAGGCTATTCAGAACCTG

47 303 AATAGAACAAAGGAAGTTTGATGTGG GAAATGACTTGCGTAGGCCA AACCCXTTAGCAAGACAAAGATTTCTG CCCTTTAGCAAGACAAAGATTTCTGGACGTGAGTCTGCCACTAAOTAGCCG

48 304 AATTGAGGATCTAGAACAGCCOC GTTTGATTCCACATGTAGAAAGGATG CTAXTCCXACCGCAGAGTGGACTTGGT AGTCCCACCGCAGAGTGGACTTGGTCTCCTGGCCAATTGCAGGCAATGAAA

49 305 CTCGTATGTCTGGGAATTAATAGTAAGAAA TCAATAACACATTTGCCTAAGTTCCT ATTTTAAATGAGTATTGATGCAATA ATTTTAAATGAGTATTGATGCAATAGTTGTTATGGAATATATTAAGGAACT

Row# REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

50 306 TAAGGTGGGCACTGAGCATG GAATGTGCCRTTTTATTACATAGGTATACA CCACGXTGGTTTGGGGXTCACCTCTTT AGCCCACCTAAGAGARGAATCATGGGAAAGAGGTGATCCCCAAACCACCGT

51 307 TACCAGAAAAGTGGGACATTCCT CTTCTGAGCCCTCCAAACTGT TGAAGATACCTGAAAATGTGGAAGC TOAAGATACCTGAAAATGTGGAAGCAGCTTTAAAATTGGGTAAGGAGCAGA

52 308 TTTGATGTTTGTGACCACTGCT CTATTGAATTGAACATATTTTATGGCTTC TGGCTTCGTGGCTTTGTGACCTTGT TTCCCACAGGTTGCTGTTTGACCACGACAAGGTCACAAAGCCACGAAGCCA

53 310 AAATATACAGACACAGTGGTTACTGGTG GTGATGGGATTATAGGCATGAGC GGGGAGGTAGAACAGAGGATACAAA GGGGAGGTAGAACAGAGGATACAAAGTAGCAGATATAAGGCACTGTGGCTC

54 311 GTGGGACCTAATTAAACTAAAGAGCTTC TGTCTGATGCACAGTTTGAAAATATT CAAAATAAACTACCAACAGAGTAAA CAAAATAAACTACCAACAGAGTAAACAGACAACCCACAGAATGGGAGGAAA

55 312 TTTTCACTCATATGGGAAAGCTAAAC CAATTAACCTTCTCCTCTCTCTCCC CTCTCCCCTCTCCCCACCATCCTTC GTAGATTGATGGTTACCAGAGGCTAGGAAGGATGGTGGGGAGAGGGGAGAG

56 313 GGCCACACATGATTCTTTACATG TGATCTGTACACATCCTGGGTTC TGCCTGGAATTGATTTTTGTATATA ATATGAGAAAAATTTAAAATCAATTGTATATACAAAAATCAATTCCAGGCA

57 314 GCCAGATGCCTTCCTTAGTAACTT GTGAAATAGTAAATAGTTAGACTAAGATGAATGCA GAATTTTGCGCTGATTATAGAGTGT GAATTTTGCGCTGATTATAGAGTGTGGAATTGATTGCATTCATCTTAGTCT

58 315 CTTGCAGTAAAGAAGAGCTTTGTAAAG TCTACTGGAGAGCGTTGTTCTGT GCTTGATGTCTTAGTTACTTTCATT GCTTGATGTCTTAGTTACTTTCATTGTTGGATCTCAGTTTTCTGAGCACAG

59 316 TTGCCACCTGGACACTTTG AGTCTGGGTCAATCACCCC CAGCCAACACTGTAACTCCTTTCTT CTCTTCCTTCCTTTAAATCAACAGGGAAGAAAGGAGTTACAGTGTTGGCTG

60 317 AGAAATTCAAGCCTGCTGCA CTCCTACAACAAAAACACACTTAAGCA TTAACTGGTTGTTTTGTAGATTTTA TTAACTGGTTGTTTTGTAGATTTTACTGTATATTGCTTTATATTGTCTGTA

61 318 TCCTTAAAAGGAAAGCTCCCAG CTGCTGGGTACACAGGGTG ATCATTAGCCAGTGTTTATTTGGCC ATCATTAGCCAGTGTTTATTTGGCCGCAGGGCAGGAGCAGGCCCTATACTG

62 319 CTTGTATGAGGAAGATTCTGGGTTAC CAGTACAGCAATAAAAACTGTTCTGG CAGGTCCACCACAGAGTCTCATTCT CTGAGTTCAGTCCAGACATAACAGCGAGAATGAGACTCTGTGGTGGACCTG

63 320 TTATTCTACCAAAAAGACATATGCACTTG CTAATAATACTGTGTGGATGTACCACATTT GAGTCAGCCTAGGTGCCCATCAACA GAGTCAGCCTAGGTGCCCATCAACAGTGGATTGGATCAATAAAATGTGGTA

64 321 AGAAGTACTGGGATGATCTTTGGG GCAAAGAGCGAAAGAACAAAGC TGATGAAAGAGAGTTGACTTGGGGG TGATGAAAGAGAGTTGACTTGGGGGGCAACCTGCTCGGGTCCCCTTCCATT

65 322 CCGGGGATCTCAAAAACCT GGAACAGGATGGATATTTTGGG TTTTTAAGTTTGTAGGACTGTTTGT GCAGTTTAGAGGTCCTTAATGAATCGACAAACAGTCCTACAAACTTAAAAA

66 323 GTGTTCCAAGAACTTTATCAGCATC AGTTTTTGTTTCCTGATTCATAGTGG AGTGGGACTAATAATACTTAACCTC TCATTATGCTGCCACTAGAACTTAAGGAGGTTAAGTATTATTAGTCCCACT

67 324 AAATCTTATTCCTCCCCCTCATG CTTATGCCTAGTTTCTTCTCCTAGTTTCA ACTATTAGTGATTCCAGACTTCATT ACTATTAGTGATTCCAGACTTCATTGTCTCCCCTAGGTGGGAATGATTTAT

68 325 TTCGTGTTTTACAAAGCATGAGC ATTCTGATGCTGCTTCAGTTTGA GGAAGCATGAGGTTGTCCAAGCAGG GGAAGCATGAGGTTGTCCAAGCAGGCCCTGGGGCCAGGACAGAAGAGAGGT

69 326 GTTCCCTGTTAGAATACACCCACC AGACCTGGTTCATACTGATTGATAGC TAATTTAAAAGGAAAAGACTCAAAC AATCAACAGGGAAATATTACACCTAAGTTTGAGTCTTTTCCTTTTAAATTA

70 327 CATATCGCTGAAGTGAAAGTCAGG CAAAAAGCTCAAGGAAATTTGG CTCXAAGXAATGGAATGGGGATATTTC CCAAGAAATGGAATGGGGATATTTCGGTTGGCTCAGATGAATCTGAGAATC

71 328 CTGGATGTGTTTCAAACAGTGAGA AGAGGGCAAGAAGGAACACAG CAGAATATTTGAGAGAGTAGGAAAA AAAGAGGCAGTATCCATGAGCCTGAATTTTCCTACTCTCTCAAATATTCTG

72 329 TGGTCTTGAACTCCTGGGC CAATAAGAAAATGAACAACTCAACTTAAAAG CAAGTGATCCTTCTGCCTCAGCCTC CAAGTGATCCTTCTGCCTCAGCCTCCCAAAGTACTGGGATTGCAGCATGAG

73 330 CTGTCTTTTTTTCTAATCAGTTTCCTTC ATTTGGCGTTTGATAATCCTCA ATAATTGATATCAGATGAACACAAA ATAATTGATATCAGATGAACACAAAGCACATAGAWACCTGATGTTTCTGAG

74 331 ATCTAATGCTTACCATGTCTTCATGG CCTTOGTATGTTCACAAACCTTTG GCCTGCCCTGTAGGACTTACCTGAC GGCTACTCAACGTCTGATTTGATAGGGTCAGGTAAGTCCTACAGGGCAGGC

75 332 TGGAGTAAGTAGGGGTTGCACA AATAAATTATTTGGATAGCCAAACTTGG AAAGGTGTTTCCACCATTGATGATT CAAGGCATCTTTACCCTTGATGATAGAATCATCAATGGTGGAAACACCTTT

76 333 CCAGGTGACCAGGCCCCA GGCAATTGATGGGCAAGTG XCTAGCAXCCCAAGCAGAGGCAGCA CCTAGCAGCCCAAGCAGAGGCAGCACTCCCTCCCCTGCAAGAACAGCCTGC

77 334 TTGGAACTGAGTAACAGGCCA CAACTATTTAACCAGTCGCCAAAA TTCACGTTCCAAACTTTCCCCGACT TTGGAAGGCTTGGAAGAAGACTCAAAAGTCGGGGAAAGTTTGGAACGTGAA

78 335 ATCTCTGCACTCTCATGTTCATTT GTGTGTGTGTGTGTGCGC CTGTCAATGTATGTTTAGTTTGTTT AGCATTACTGTAATAGCCAAATATGGAAACAAACTAAACATACATTGACAG

79 336 GTAATATCATTAGGCACCCAACTAACAT GGAGAGATGTGAGGTTTGAATTTC GTCAGAACTGTATGTTGATACTTGC GTCAGAACTGTATGTTGATACTTGCCTTCAGACAGTAAAACTAATTTGAAA

80 337 ACTGCCAGTGGGTTGTTGAA TAAAGGGTAAAATCCAACAAGAACAC TTGATGTTCCAGTGTTGGGTGCATA TTGATGTTCCAGTGTTGGGTGCATAGAAATTTAGGTATTTAGGCTAGTTAA

81 338 CCGGGGATCTGACTATCTTGT CATAACATGGACACTGAGTATAATATCTCC TACAGTCCATCAGTGAAATAATATG ATACATATTTAGGGACTTTTAACACGCATATTATTTCACTGATGGACTGTA

82 339 ATCTAGAAATAGAACAGCCTGAATCTGTAA GAATGGGAGTACTAGCCATCACC ACTGGATCTCTATCTTCTXAACAGCCT GTCAGTTGACAGCAGCTGCATTACGAAGGCTGTTAAGAAGATAGAGATCCA

83 340 AGTGAGTGAGGTGCGCTACAG CAATGGCAGTATTGGTGTTATGC ACTTGACTGCAGGTTCATTGAGGGC AACACAGTTGAAAAGAATCTGACTTCGCCCTCAATGAACCTGCAGTCAAGT

84 341 CGGGGATCTTTCTTAAAACCAT GTAAAATGATTAGATGTACTATTTAAGGAGTGACT CTCCGAAATAAGCAGAAAACTAACA TATCTATGATAATTTGGAGACAAACGTGTTAGTTTTCTGCTTATTTCGGAG

85 342 CAACATGGTGAAACCCCATC CACCTCCCAGGTTCAAGAGAT AAAATTAGCTTGGCGTAGTGGTGGG AAAATTAGCTTGGCGTAGTGGTGGGCGCCTGTAATCCCAGCTACTCAGGAG

86 343 TCACTAAGACAGTCACAGTCAATTAAA TTGGTTCAGTATTATAAATTGTTATTGGAAG ACTTAAAAAAAGAGAAACAAAAGGA ACTTAAAAAAAGAGAAACAAAAGGACGTGTCAGGACAGCTTCCAATAACAA

87 344 TCCCATGCTCTCTGTTTCC GGGTTTGGGACTCCAGGA CTGGGAAGCCAAGXTCATCAAGATCAT GGGAAGCCAAGATCATCAAGATCATAGTCTTCTTTTGCTCACAGCACACCC

88 345 TTATATAAAATTCTGGATACAGGCTCCC CCTACCACTTTCCTCATAAGCCA GCAGGCCTTGAAAATGGAAGTTTAC GCAGGCCTTGAAAATGGAAGTTTACCATTTTCAACGGAAAGAAGGCTGGCT

89 346 CTGAATAGAGATTGGGGAAAAAGAA TTATATAACTCTTTCTCATTTTTCTGGCTTT GGCAATTGGGGGTGGTCTGGAGAGG GGCAATTGGGGGTGGTCTGGAGAGGGTACATTTAAACCAAAGCCAGAAAAA

90 347 GCCCTGGGAAGAAAGACAAC TTTTTCCCCAATCTCTATTCAGTTAC TCAGTTACCCTTTCTTATTTCTTCA GTAATTTTAGTTTGTGGTAAGTACAGTGAAGAAATAAGAAAGGGTAACTGA

91 348 AAACTGTTCTTGGACTCAGGCA CATTTAATCTGCATGACAATCCC AGCCTGTGCCAGTCTXAAACTTGCCTA ACCCAACACATAATCTGGCACCTTCCTAGGCAAGTTTAAGACTGGCACAGG

92 349 GAGGACACATACTGTCATTGGATG TGTACTTAGCATACAAATCTCTAGGATGATT ACAAGCCAAATATGTTACTAGAAGT ACAAGCCAAATATGTTACTAGAAGTAATTAATCTATTCATTCAAATCATCC

93 350 CCRTATTTCTAACAGGAAAATAGTCATTTTA AGCTCCCCATACCTGTTTGC AGCTGTATAATAGAGTGTCTCATAC TAAAGGTCTGATTTTTTACAAAGTGCGTATGAGACACTCTATTATACAGCT

94 351 GAAACTAATGAGAACAAACACACAACAT TTTTGATGTGAACATTTAGTGCTATAA GCTATAAATTTCTCTCCTAACACTG CCAGAATCTCTGGGACACAGCTAAGGCAGTGTTAGGAGAGAAATTTATAGC

95 352 GTGAAGATCGAATACCTAAAATTATCTTATTTAC AGGTTTTGTTCCAACTCCAAGG TGGAGATTAACTCTTCACTGACTTC ACATAGGTTAAGTTCATGCTTTCAGGGAAGTCAGTGAAGAGTTAATCTCCA

96 353 GCGGGATCTCTACAAGGAGAA GTATTTTGATGGGAATTGCATTG TKTTTCCTTTTTTGTGTGTTTCATC ACAMACCACTGCTGAAAGAAATCACCGATGAAACACACAAAAAAGGAAAMA

97 354 GCTGAAAGAAATCACYGATGAAAC TGTATGCATTGCTTTGGGC CAATATGGTCCTTTTCACAATGATT AACATCCCATGCTCATGAATTGGAAGAATCATTGTGAAAAGGIACCATATTG

98 355 TCTGCTCACTCCAATTCTGGG GATTCCAGGCTGTCAGCTCC CGAAAATTATTTCAGTCACATTGTT CGAAAATTATTTCAGTCACATTGTTCGTAGCAACCATATTTTAAAGCACAG

Ro # REF# Upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

99 356 TGAGGCAGGAGAATCACTTGA TTTTTTTTTTGAGATGGAGTCTTGC ACCCAGGAGGTGGAGGTTXCAGTGAGC CCAGGAGGTGGAGGTTGCAGTGAGCAGAATTCACACCACTTCATTCCAGCC

100 357 AATGGGCGCACTGCTGAG P CCTCATTTGTAAAACTGGCAAGTAA CAGAACCCAATGCATAGTGTTATTC ATAGGAATCTCTTCAATTAGTCCTCGGAATAACACTATGCATTGGGTTCTG

101 358 TTAGGGCAAGGATAAGCCAGTAT P GTCTATAGCAGGTCAAATAWCTAGGCC GCCTAATCTTAGCCAGAGGGACCAG GCTGTATTTGTTCCTTGCTGAGGGCCCTGGTCCCTCTGGCTAAGATTAGGC

102 359 TTGGCTTACTGCAATCTCTACCTC P GACCAACATGGAGAAACCCC CCATCTCCACTAAAAAAAAAAAAAA

103 360 TTAAATGTTTATTGGCTGTCTTCACC P AAATGTAGACATGCATTCACTTAGGAA ACTTAGGAAATATTTATGAACATGA CCCTATCAGAACTCCCAGCATGTAGGTCATGTTCATAAATATTTCCTAAGT

104 361 TAATATCCTGATGAATGAAGATGGCTA P CAATTAGCTCTGAAGAGTCAGCCT CCAGATACTGACCTCCTATGAAAGT CCTCATACCAGAAGATGCCATCAAGGACTTTCATAGGAGGTCAGTATCTGG

105 363 GAATTATTTTCTTTGCACTGTTAGGACA P CATAGTCATTTGGGGAACTGATATTG TTGTTXGAGXTTAAAAATCCAGCAAAA ATCAAATGCCTAACAATTGGTAGTACTTTTGCTGGATTTTTAATCTCCAAC

106 364 TGCAAAGAAGATTTTTGAATCCC P GACTGGGTAATTTATGAAGAAAAGAGTTT CAGGAAGCATTACTGGGAGGCATCA TCACCTTCGGCCATCATTGTGTGTCCTGATGCCTCCCAGTAATGCTTCCTG

107 365 ACCACCATGTCTGGTTAAGTTTTTAA P CATAATTCCAGCAACTCAGGAGG GGCTGAGGCAGGAGGATAGCTTGAT TCCCAAGGTAGTCTCAAATTCTTGGGATCAAGCTATCCTCCTGCCTCAGCC

108 366 TACGTAATCATTATTTTTCACAGGCTG P GTCTTGGTCTCAAAGTAACCCAAA CXCAAAXCTTCCACCTGCCAAGGATGG GGTGGCGGGATTAGCCTGACGCCACCCCATCCTTGGCAGGTGGAAGGTTTG

109 367 CTAGGCATGTCAAGTTCTTCATGG P CTTTATTCCTCCCTACCCTCATTG CTAATGATGAGGGCCAXTTCTCCTTGA AGGAGAGAAGAGGGGTCTTCATTTTGTCAAGGAGAATTGGCCCTCATCATT

110 368 AAAATGCCCTOTCTAGAGGGAG CTAATCTCATTCCTCCTTTCTTCCTT GCGCTGTGGGAATGTAGACTTCTTG GCGCTGTGGGAATGTAGACTTCTTGGGGAAGATTTCCGAGTTGAGTAAGAA

111 369 TCCTCATTACATTTTAATGGCACTG TGCCCTGTCTCACCTGCA AGTTACAAAATGAAAAAGACTAAAT AGTTACAAAATGAAAAAGACTAAATGTTAGTAGAGTTCAGAAATCTGTGGC

112 370 TTGTGAGGCAACCCCAGTATAC P CTATACAGTGCTACTCTACTCACACCCA AGCTCCCCACCCGCACACACACACA AGTTTCTTGATGACTTTGAGTGTCAGTGTGTGTGTGTGCGGGTGGGGAGCT

113 371 TATGGWAGTGAAAGCAGAGTCTGG P CCAGATATGACYCCCCTCC TCCCCTGCTCAACXACTTGTTATGGCT TCCATTTTAACCCAAAGGCAATGGGGAGCCATAACAAGTGGTTGAGCAGGG

114 372 CAGTTGTTGGAAACACTATTATTTAATGTACTAT GCAGAAGGATCTAACAGATGAATTTG CCAATGAATTTACGGCACACTATCC CCAATGAATTTACGGCACACTATCCGCTCTAATCTAAATTTTTGTGTATCC

115 373 CGGGGATCTGAAAAAAAAATTC AGAGCCAGCATTGTCTCACTTG ACTTTATAAATGATTTCACTGCATC ACTTTATAAATGATTTCACTGCATCCCCCAAAATTCAATCCCTAATTTACA

116 374 TCCCAGATAGTTCCAGAGACTTCA P CTTTAGCTTCAAGTTCAGAAGATGACTT CTTTTTACTATGTTTTTGTCCTCCC ATGAGCACATGTGAGTGTATATGGGAGGGAGGACAAAAACATAGTAAAAAG

117 375 TCACAGAAAGATACTGGAGAGGGT CAAACCCCTAAGCCCACCTA ACTTTGGGAGGAGTTTGGGGCTGTG ACTTTGGGAGGAGTTTGGGGCTGTGCGAGGGTGACTCATTTTTACCTATGT

118 376 AGACTTGTTGATGGCTTTGACC CTAGGGCAAAATGCCACTAGTTT GATGAGGAACTTGTTGGGAACTGGA GATGAGGAACTTGTTGGGAACTGGAGTAAAAGTCACTCTTGCTAGGCAAAG

119 377 TATCAOTCTCTTGAGCTATGGGG P CCGAGGAGGGACCTTCAA AGAGCTCCAXATCCAGCCTCATTTCAC GGTTCCAGGGCTCAGCTTCTTCATCCGTGAAATGAGGCTGGATTTGGAGCT

120 378 CTCCTGGTGTGGGCTTCTG P ATCTTGGCATTGAGAACAAGAAGA GAAGAACCAGGAGCCAAGGTGTGTA AATGGCAGAGGGAAACCTCACCCAGGTACACACCTTGGCTCCTGGTTCTTC

121 379 CAGATTCACAGACTAGAAAGGGAGTT P ATCGAATTGAGAAGTAAATCGTGTG TCGTGTGCATGTACCTGTGTGTGTG TCAGTAAAGTTAACCACACTCACACGCACACACACAGGTACATGCACACGA

122 381 TTTTTRGTTTGAGGCTTTTATAGCTG GCAACGCTGAGGGGAAAA GCTAAAGATGTGCCTCAAGACACCC GCTAAAGATGTGCCTCAAGACACCCCGATGTTAGATGCCTTTCCCAACACA

123 382 AGATGATGTGAGCTAAGOGAAGTCT P CTCTGTACTTATTATTCTCTCTACCTGGGA TAATCTCTCTGCCCTATTCTCTTTC GAAGATGTGTAGAAACTACACAAGAGGAAAGAGAATAGGGCAGAGAGATTA

124 383 AAGACAGGGAATGTTTCTCTTATTTCTC P GTGAGTCATGTCCAGATGAAAATTT GAATTAXCAGACTCTTGCTXCATCCAG GAGTTCTGGTCTTAATTATGCCTCACCTGGATGCAGCAAGAGTCTGCTAAT

125 384 CATGGAGACACCGGGTGG P CGAGAGATCTTTCAATTTCTGTTTG AATGACTACTGTATCCAGGCTTGAC ATGGGCTGCAGGCATACATTCTGAACGTCAAGCCTGGATACAGTAGTCATT

126 385 GGGGATCTCAAAAAAGAAAAAAAA P GATCTCAGCTCACTATATCCTCTGC AGTGATTCTCCTACCTCAGCTTCCC GCATGTACCTGTAATCCCAGCTACTCGGGAAGCTGAGGTAGGAGAATCACT

127 386 TATTTCCCCAAATTATCRTAACTCCA P CAAAAGAAAGAGTGCAGCGG CCCTTTACAAGCCAACTCTTCGTCT CTTCTGTGGATTCCAGAGAGTAAGAGAGACGAAGAGTTGGCTTGTAAAGGG

128 387 CGGGGATCAGCCATTCAA TGAAAGTGGTCCTATCCACTGTG ATTGAAAATATTATTATGTGAGGGC ATTGAAAATATTATTATGTGAGGGCCGAAAAAGATGCAATCTGTACTCTCA

129 388 ATACTGTGAAAGATATGAGACTCTTGGG P CTTAAGAACTGGTCAAGAAAATCATGA AAATCATGACACTCTGGCTATACTG GGATGGCGTGATACTCACAGCAACGGCAGTATAGCCAGAGTGTCATGATTT

130 389 ACACAGTGGGCMTGCTGT P AACAGAAAGGAACTCAGAATAAGCC GTATTAAGTXTAXATAGATTTTTTTTT AATCCCAGGCTTTACTGGCAATAGTGAAAAAAAAATCTATTTAGACTTAAT

131 390 TTCGTGATACTTATTTAAAAGCATCTTTG P AGACATAGGACACAGACTGAATTTCC AAAAATAATACGXTACACCAATATCAA GGTGATAAAAACATTAAGAAATATTATTGATATTGGTGTATCGTATTATTT

132 391 TATCGTATTATTTTTCCAGGGGATTC P ATTCAACAGTGAAGACAACTTTCACC ATGCTTATCTCTTGACTTCTTAAAA GGATTTCTGAATATATTTTTAAATGGTTTTAAGAAGTCAAGAGATAAGCAT

133 392 AGTAGCTAAGACTACAGGCATGTGC CTAATACCAGTGCTTTGGGAGG TTTTGTAGAGAAAGGGTCTTGCTAT TTTTGTAGAGAAAGGGTCTTGCTATGTTGCCCAGGCTTGTTTCAAACTCCT

134 393 GTGACTCTTAGTGTGTAACAAAGTGCA P TACCTGCATAATAGAGCTTCAGTAAAAAC CXTCXGTAAAAACTATGGACACTGAAG AGTATTACCATAGAAGCTTGCCTGAGCTTCAGTGTCCATAGTTTTTACTGA

135 394 CCCAAGTGTTACCCAAAACATACTAA P TCATGGATGCTGGGAGTTG GGTGTTCCTAGTTAYGGTTGCCAGT GGCATTCTTATGTTTTTATTTGCTAGACTGGCAACCRTAACTAGGAACACC

136 395 AATGTCAAATTTAAGTGGGCATTCT P CTTCTTTGGCTTTGAGTCTGCA GCTGGGAGTTGGGTGTTCCTAGTTA GTTTTTATTTGCTARACTGGCAACCGTAACTAGGAACACCCAACTCCCAGC

137 396 AATGGGAATCTTAGTGAGTTTGTTTCT GATTGGTTTCCAGAGGCATATTG TCTCTAACTGGGCCATCTAATCCTG TCTCTAACTGGGCCATCTAATCCTGCGCTTCCAGGTGTTCCTGGGGAGAGG

138 397 TTCGCAAGAGTGAGGGCTC P ATTTTCTTCCTCACACCTGATGC ATATACTTTCTGCCCCCCTCCACTA GGCAATGAGTTTGGCTTGCTGAGAGGTAGTGGAGGGGGGCAGAAAGTATAT

139 398 CTGTGTCACAGGCATTTGAGG P CGGTAACGGGGATCTGATG AAACCCTTCATGXTAAAAATCTAGCCC CACACAKGAAAAGAGAGCTGAGATTGGGGCTAGATTTTTAGCATGAAGGGT

140 399 TCGGTACACCAGGGGATCT P CATTTAAGAACACCTGAGTTATGCTACA AAAGAAGATXCTATCTGTGAGAAAGCA AATGACTTAAAAATTTTTTCTTCCTCTGCTTTCTCACAGATAGCATCTTCT

141 400 AGCTTACATTTCTGCATCAGTTCAT CCTTTTCAGACGGGCACTT TCTTGGATCCATCCTCTGAAAGGTC TCTTGGATCCATCCTCTGAAAGGTCGTTTCTTCTCCATAACACTCCTTGGG

142 401 ATGAGAACCCTGGAGGTATAGCA P CAATTTTGTCTATACATAATCTACCAAGCC CAAGCXAGTGATTTXCCTGGAATTATC STAGTAATTGACTCTGAAACCCAAGCGATAATTCCAGGAAAATCACTGGCT

143 402 ATTATAAAGTCAGAGAGGTAGAAGGAGAACA P CTTTTGTCTGTGCTGCTCCTAACT AGTCCACTGTGCTCGGCTGTCTACT AAATGAGGTGCTTTAGAAGTTACATGAGTAGACAGCCGAGCACAGTGGACT

144 404 CTTGATAGGCGGCTTTTACCTT P GACATAAAAATTGGATAGAAAGCATTTG AAGCATTTGATXTACAAGATTAAACTG TCTGGTTGCATTGCAAGGTGGCCTCGCAGTTTAATCTTGTAAATCAAATGC

145 405 GTGGATACAGTTCAGATTCCTAACCA P TTATCACATGGGGACTACACCC ACCCCATCACAGTCTCATCTTATTC TGGGAAAAAGTAAAAGATATGTAATGGAATAAGATGAGACTGTGATGGGGT

146 406 ATAGTGCACAGTCTTTATCCCTTTTTT P CTTTGTGGAGGAGGTAACTTCACA CAAATGGCTTXCTTTGGAGGCTTTATG ACCCTGTGGACTGGAGCTAGGGGTGGCATAAAGCCTCCAAAGGAAGCCATT

147 408 GAACTGCAAAATCTCTAATATCTTATTTTCTG GGGGGAAGGGATGCTAATTAA CCTCTGAGAAATGGGGATATGATGC CCTCTGAGAAATGGGGATATGATGCCTATTAAGCTGAGATAATCAAAAATA

Row# REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

148 409 CCRCTGAATTTATATAGTACTTTTTTTTGG TCCACCCAGTTCGAGCTTC CGGCCGCTTTGTTTACCAACTGTTA AAACTTTCCTATTATACATATAATACTAACAGTTGGTAAACAAAGCGGCCG

149 410 GGAGCTTGCAGTGAGCTGA CATGAGGAACTGTTTTCTTTTCTTTTT TTTTTTGAXACGGXGTCTCCCTCTGTC ATCGSCCACTGCACTCCAGCCTGGGCGACAGAGGGAGACTCCGTCTCAAAA

150 411 AGAGATTAGTTGCTGGGCTTTG GCTTGCAGACTTGTCCAGC CCACAAATTCCAAATAACAGAACCA GTTGCAGGATATATTCCTTTGTTGGGTGGTTCTGTTATTTGGAATTTGTGG

151 412 TGAAGGAAAAAAAAAAAAAAGGC AAGCATATGGTCTGTCCTGGAG TGACACTCTACCCAACAGCAAAAAA TGACACTCTACCCAACAGCAAAAAAACCTTTCTTCTCAAGTATACGTGGAA

152 413 TGTGGATGTACCACATTTTATTGATC GTATATGACTCAAAGAAAGTCAGTTATTCTACG GGGCACCTAGGCTGACTCCXTGTCTTT GCACCTAGGCTGACTCCATGTCTTTGCTATTGTAATAGTGCTGCGATGAAC

153 415 TTTTGCACATTGAGTGCTTAGG GATTAGAAACCACCATGTTTACAGGA AAGCCCTXAATCCAGAGAXATGTTTAC GCCCTAAATCCAGAGATATGTTTACCACTTCCTGGCCCTGCCTTCYTAATA

154 416 ATAGACCTAAAGCCCTAAATCCAGAG GTAAAACATTCCTCCAAGAACAGGT GGACCTATCCAGAAGGAGATTATTA TTTACYACTTCCTGGCCCTGCCTTCCTAATAATCTCCTTCTGGATAGGTCC

155 417 AATATTTCCAGGAAGCTGCATTC CTTATCATATTTTTCTCTTTACCATTCTCTTAC GGTAAGGGGAGCTACTGGTCATACA GGTAAGGGGAGCTACTGGTCATACAGCAGCATGGAGTGAGTGAAGTGAGAG

156 418 TCCTCCAGGACAACAAGGATT CATATAGGAGTTTATCTAAGGGAAATCACA TATGCCCTACAACTGCTATAACGCT GTATTTGCATGCATGCAAAGAGTGGCAGCGTTATAGCAGTTGTAGGGCATA

157 419 ATCATGGCCTGATTGGCA CAGAGCCATCAGTTCTCCG GGGGAAGCAGAGGGAAGACAGTGGC GATCTTCCCCCAGGCTCTTGGTGCACGCCACTGTCTTCCCTCTGCTTCCCC

158 420 AAGAGAACAGGCTCCTTTTCAAA GTATTTTTTGTAGAGTGGAGGTTTTGC AGGTCTGTGATGGGGTAGAAAGCAA AGGTCTGTGATGGGGTAGAAAGCAAGAGTTCAGCCTGGCCAACATGGCAAA

159 421 GAAGGCTGAATGATATTCCATTGT AGAGAGACTGCGGGACCAT GTGGTAGTTCTTTCTTTAXTTTTTTGA TTTATGGAAAACAGTATGGACGTTCCTCAAAAAATTAAAGAAAGAACTACC

160 422 CAGCCCAAAACATTCAAGGA ATTTCGATGACCACTTACCCTTACA CAAATCATTATTATTTTTTTTATAGCTCACAGTGGCTGTCTGTGCTTTTGT

161 423 TGCCCTCTTCCTGTAACACC CTGACTCCTTTTGAGTATCAGGGA CAGGGAACAAGCCAGRCTCTGTAGA GTGAGAACTTCTTGTGCATTAACACGTCTACAGAGYCTGGCTTGTTCCCTG

162 424 TGCATATAGCAGATCCTGAGGC AGATGATATTTGGGTGAGGACACA GGACACAAATCAXAACCAXATTATTCA GTCTTCTAAATAATACATATTGTCACTGAATAATATGGTTATGATTTGTGT

163 425 GTTATGTCATGTGCTGTAAAGTTAGGTCT ACTTCAAAAATTCACATAGCACAATTG TTAAAAATXAAAAATTTAGATTAAAAG AAAAATAAAAAATTTAGATTAAAAGCTAAATTTAAGAAAGTTACTTTTGAA

164 426 TAGTTCAAGACCAGCCTGACG GTTTCAAGCAATTCTCCTGCC CCCAAATAGCTGGGATTACAGGAAT ACAAAATTAGCCGGGCGTGGTGGCGCATTCCTGTAATCCCAGCTATTTGGG

165 427 GCAAAGTATCCCAGACATGTAAACA TCCTGCTATTCTTCAGCTAATTTGTT TGATAATTTAAAACAATAACAAGAG TTTTCAACAGTACTAAAAGAAAACAGCTCTTGTTATTGTTTTAAATTATCA

166 428 TGTAAATCAATKTATTTTCAGTCCAGG TTTACATTAGTTGCTTTCCTCAGGTG CTCAGGTGCACACATCATTCTCTCC CTGCTCAACTACCATTATCCAGCAACGGAGAGAATGATGTGTGCACCTGAG

167 429 TGTTCTAGTTAGAAGAATTTACACTAGGAAAAA CCTCAGGTGCACACATCATTC AATGGTAAGAATAGTGTAAATCAAT AATGGTAAGAATAGTGTAAATCAATGTATTTTCAGTCCAGGGAAGGCTGCT

168 430 CTCTGATATTTACACTGACGATTTGCT TCCTGAGRCCTCCCCAGC ATACAAGXTTAAAAGAGAAATCGTTGA

169 431 CAAAACACTTCTATACCTAATTCCCTTTT TKACCTTGATTTATACATGCTACGATC TCTCCATGAAAAGATTXACTGTTAAAC TCCATGAAAAGATTAACTGTTAAACGCCCTTTCTGCATCTGATGATCGTAG

170 432 GGGGATCTTGGGACGCTG AATTGTTGGCTTGAAAGGTAATTCTAT ATTCTATTTTAAAYTCTTTGTGAAA AGCCACTGTTGAAAGCTGTTTGGAAGTTTCACAAAGARTTTAAAATAGAAT

171 433 TATTATTAGGCCATTCTTGCATTGC CACCATGCAGATGCCGTC AGGAAAAGAGATTTAXTTGGCTCACAG GAAAAGAGATTTAATTGGCTCACAGGTCTGCAGGCTGTACAGGAAGCATOG

172 434 GGTACCGGGGATCTTGGG TCTTTGTGAAAYTTCCAAACAGCT GGCTXAACTAATTTACATTCACACCAA GGAGAAAAGGGAACATTAATACATAGTTGGTGTGAATGTAAATTAGTTCAO

173 435 TGGAGGTTTTGTTCCCAAAC TAAAATGCGCTGGAAGCTCTC CTCAGCAGTAGAATCAAACAAGTAG TCTTGGAGGCTCTGAAATTCTTTGTCCTACTTGTTTGATTCTACTGCTGAG

174 436 GAAACTGCCCCCATGAGC GTGTTCCCACCCAAATCTCA CAATCACYTCCCACCAXGTCCCTOCCT ATCACYTCCCACCAGGTCCCTCCCTCGGCACATGCGGATTACARTTCGAGG

175 437 GGCTTGCATTCCTAGTAAAATTGA TTAGCTTTCTGAAAACTCATTATGTTGA ATTATGTTGAATATGGCATTTGGGA GAGCCTGTATCCCTTACAGTAGAAACTCCCAAATGCCATATTCAACATAAT

176 438 TATATCAGTGGGCTCCTTCCTTG CTTTATAGCAGTATGAAAATGGAATAATACACT CCTTTAAAACTACTGAAGTCTGAGC CCTTTAAAACTACTGAAGTCTGAGCCTGACACCAGAGATTCTGATTTGCTT

177 439 CATGTGATAGAGTTATGGCTAGTGAATG TTTTAGAATCTTTTCCATTTATCCAGTG TATCCAGTGAATGGGGAAAAAGAGA AAAATCTTCCACAAAATTCTTTGCACTCTCTTTTTCCCCATTCACTGGATA

178 440 TGATGAAAAAAGGCACATGTCA GCCTCAAGTTTCAGGGCC TCTTTGCATGGGCTGTGGGAGAGGA ATATGGCCATTGAATACCTTGCAAAGTCCTCTCCCACAGCCCATGCAAAGA

179 441 GCTAGACATTTTCAATAGGCACCTC TGTAGCCCAAGGTGGTCAGA AAATATAXATTTTATTTGACATCTCCC AAAAAAGTTTCCAAAATTTAAAAATGGGGAGATGTCAAATAAAATCTATAT

180 443 CAACCATTTTTCTGATAATTCTGTGA AGGATGTCACCATTTTTGTGGT TTTTTTGCAATAATCCATTAGAAAG GTTTTATCTTTTCTTAAAAATGTGTGCTTTCTAATGGATTATTGCAAAAAA

181 444 TRCTCCTTGATCCGTGGAGT TGCACCTAGTTACCCAAGAGCT AAGATGTWCAAGATCAATGTTTTCA CAGAATGGATGTTGTATTAGCAGACGTGAAAACATTGATCTTGWACATCTT

182 445 TTTGCTTCCCCTTCTGCC CAATTTACTGTATTAGCCTGTTCTCACA GAGTGGGCAATTTACAAAGGAAAGA CTATGGAACTGTGAGTCTATTAAATATCTTTCCTTTGTAAATTGCCCACTC

183 446 AGAAGTCTTTTACATGGAAGAAGAAAAAA CRAAGACTAACAAAGGTGGTCTAAGG GTCTAAGGGAACAACCGAGTGTACC CTAAGAAAGGAGAGGTTTCAATAGACGGTACACTCGGTTGTTCCCTTAGAC

184 447 AGGAAAGTCTTTGACTCCATCCTC AACCCCAGCTATTCTTATTAGCACA CTCATACTCAGACCTAXTTCCTTCTAA CATACTCAGACCTAATTCCTTCTAACAAAGGTTCTTTGGAAGGTATGTGCT

185 449 CCCACCTCTAGCAATGCAG CAAATCAAAACCCCAATGAGATAC TTACAATTCAACATGAGATTTGGGC TTACAATTCAACATGAGATTTGGGCGGGGACAAATACCCAAACTATATGAA

186 450 CTGGGGCCAACTGCATTT AAGCTGGTGGTAGTAGAAATACATGG CCACCTGGATTCCACAAGACACCCT CCACCTGGATTCCACAAGACACCCTGGCATCTTTATAATAAAATCCATGTA

187 451 AAACAAGAAGACAAATGGAGGTTG GTTTCTGTGGAGAGGACCGAC AACCAGTTAGAGTTCCATTTTGTGC AACCAGTTAGAGTTCCATTTTGTGCCAGGACAGTTCTGCCAAAAAGAACTT

188 452 CAGAATTGCATTGGCTATTTGG TCCCCATGTTCGTGGATTAG AATXTCTTTTGTATTCGGATAGTGA AATATCTTTTGTATTCGGATAGTGACTTAATTCTGTAGCTCTCTTTTGGTA

189 453 GTTCAACTACTTCCCACCGG TTTCAGGTTTCAATGTGTACTTTTAACA TATTTATAGATTAXATGTATTTTATTA ACCATATCACCTGTATTTAATTTATGTAATAAAATACATTTAATCTATAAA

190 454 TAGAAACTGGAAAAAGAGGGAGC GATACAGASAAGATTGCAGTGTGG GATGCAGTCAAGGCAAGGAAGAGGG GATGCAGTCAAGGCAAGGAAGAGGGGGAAATACCCTTCTTCTTGCTTTCCA

191 455 TCATGGCTGGAAATGAAAGTATC CTGGAATGGGAGAATAGTGTGTTT CAGTCACAAAGCAGAAGACCAACTC TGAAGTTCCTGAAACATTACTTTACCGAGTTGGTCTTCTGCTTTGTGACTG

192 456 AAACCCCGCCGAAGGTAC TTATTCATTTTTTTCCCCAGCAC GAGGATCTGCTGCACCAGGCAAATT GAGGATCTGCTGCACCAGGCAAATTCTGTGCAAAATGCAAATTTCTGTGCT

193 457 AAGTGGGAGGATCACTTGAGC CTTCTTTGTCTTTTGTGAGACAGTGT GGXTXCAGTGAGCTGTGATAGCACCAC CTGCAGTGAGCTGTGATAGCACCACAGAACTGCAGCCTGGGTAGCAGAGTG

194 458 ATGGTGAAACCCATCTCTACTAAAAATAC CTAGGTTCAAGCGATTCTCCTG GGCGTGGTGGTGCACTCCTGTAATC GGCGTGGTGGTGCACTCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGG

195 459 CTGCCTTCCTCTTTGCCTTC TTATAAAGAACAGAGGTTTAATTGGCTC GTAAGTTTTCTGAGTGCCCCCCTCC GTAAGTTTTCTGAGTGCCCCCCTCCCACCTTGATCATGTTTCCTATACAGC

196 460 CGTAGAGTGAAAAGGAGTGGGA CTGGCTTGGGACGAAATTTT TTGAATGAATTTTTTTTAGCATAAA TGAAAGTATAAAACCCTTAAAAGACCTTTATGCTAAAAAAAATTCATTCAA

Row* REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

197 461 AGCTGCCTTCTTGCTGTGTC GAACTGATAGGATTAGTGTCTTTATAAGAAGAGA AGAGGGCTGGCACTTTCTTTCTGTG CACAAGGCTTTTCCTGAGTTCCTGCGCACAGAAAGAAAGTGCCAGCCCTCT

198 462 CTGGGGGCTTTGTTTTGTTT AGTGACCATATCACCCATACAATCTATAG AGTTAATGCAXTTCCCATCAAATTA TTTTAATTTTGTGRAAATAACGTTGGTAATTTGATGGGAATTGCATTAACT

199 463 AGCTGGTATCCTATGGGCTTTT CCCAAGACAGCAGCCATG CCAGCAGAACTGAGAAGAGGAAGGT CCAGCAGAACTGAGAAGAGGAAGGTGGGGGAGCCCAGCTTCTCGGTGACTG

200 464 GTTGAGTTCAATTATTTTTCTTTTCCAC TGGTAAATCAGAGACTGTAGTTATCCATC AATTGGGTCTAATTTGATAAACTCC AATTGGGTCTAATTTGATAAACTCCGTTTCCAGCGGTTAAAATATAGATGG

201 465 AGCTTGGGTTCAAGAGGGAG TGGCCTGAAGCCCACGTC TCGXAATGCGCTTCAGAGGCTTTTCCT TCCAGCAGGCGTAAGTAGTGGCTGCCAGGAAAAGCCTCTGAAGCGCATTCC

202 466 TCCTGGGTTCAAGTGATCCTC CAACATATGGAGATCCTATCTCTACAAAA AAAAAAACCCAAAXTTAGCCAGCCA GCTGGGACTACAGGCTCGAACCACCGTGGCTGGCTAATTTTGGGTTTTTTT

203 468 ATAGAAATGATTAGACATCAGGAAGAATTC AGCTAATTTTACACCTAGATAGCTGGAGA GAGATGTGGTTAATAAGAAGAAACA TTTTCCTTCAAATGTTATTCAAAATGTGTTTCTTCTTATTAACCACATCTC

204 469 CACTTTACTTAGTATTTCAAAAACTTCTGCA GTTTATACTGATGCCATTATATTCCTGATC ATTGXGAAGAATGAATCTCTCACTAGG TGTGAAGAATGAATCTCTCACTAGGAAGTTGCATTAGAGGATCAGGAATAT

205 470 TTTTTTTGGTCATATAAAGCTAATGGG GTACTTTTAAATGAAAGGCTGTGGACT TGTGGACTCCCATGAACCTAGAATG ATTTTTTCAATCTCAGGATCACACAGCATTCTAGGTTCATGGGAGTCCACA

206 471 CAGCCTCCAAAAAGACAAAGTC TCTGTAGCCAGCAAGGACTGTAT ATTAAGAAAAAAAAAAAAAGATCAG AAAATGCTTACCCAAGTCAATGAGCACTGATCTTTTTTTTTTTTTCTTAAT

207 472 AAAAAAATAGCTGGCATCCAA GCCAACCTCGTCTGAGCTC GCTGACCCAGCCCCCTTTGTCTCCC GCTGACCCAGCCCCCTTTGTCTCCCGCCTGGGTCCCAGAGGCAGCAGAGCT

208 473 ATTTCTGTCCTATCAGAATGTCCTTTT GATTGTAAAATGCACCAATCAGC TAAAACGCACCAATCAGCAGAATCC ATCTTCCCTGTGACTGGCTACTTTTGGGATTCTGCTGATTGGTGCGTTTTA

209 474 TTAGTGGACTGAAGTTATAGGTGCTAAGA TTCTAAAATGCTTCACATCCCC CATTXACTTTTTCTGTAACTTAGGAGA TTTACTTTTTCTGTAACTTAGGAGAGTAGGTCACCCATCAGCAAAAGGTAA

210 475 GAAGGCAGAGTAGGTTTTTTTAATTCA TATGGGCCTGTATTTCTGATATTTCA TAATTATTTTCCTTGGCAGGGATTT TAATTATTTTCCTTGGCAGGGATTTGAAACATTATTTGGCAACTGAAATAT

211 477 GACAAGTTTTGGCTGTTGATCTTC CCAAATCTTCCAAAGTAGGCAA CAAAGATACAAATTTTCAGACATTG AAATCAATCTAAGTCTGGCATTTACGCAATGTCTGAAAATTTGTATCTTTG

212 478 TTAGTTCACAGTACTCGATAACCCTTG GCCCTTCCACTCTACACACAC CACCGTCGGTAGCTTCCTCATGCCT GCACTCGGTTGAATACTAATTAATACAGGCATGAGGAAGCTACCGACGGTG

213 479 TCCACAGCAAATAATGCATGAA GTTTCATAACTGATTTCTTTTTCCAGTG CCTTGAAACTACTGAACTCATTAGT CCTTGAAACTACTGAACTCATTAGTAGCCGGAGCAATAGACTGAGTGCAAA

214 481 TTTTTTTTTTTTAAAGACAAGTCTCGC ATTGCTTGAACCCAGGAGG GCGGAGGTTGCAGTGAXCTAAGATCAC TGTCCGCCAGGCTGGAGTGCGGTGGCGTGATCTTAGCTCACTGCAACCTCC

215 482 TTTACAACTCAGAAAGGGAAACAAG CTATAATGCCCCACCTCTGTTCT TCCCCTCCTCTGGTTTTTTGTTTTT TCCCCTCCTCTGGTTTTTTGTTTTTGGTTTTTTTTTTTTAACTGAGTCATG

216 483 CTAACCAACAGCCAATAAGAGAAAAT TGCATAAAGGTGAACTCTTCTTGC AACATTTGGAATTTTCTTACCTTTA AACATTTGGAATTTTCTTACCTTTACGCAGACTTTTCACTGCTCAGAGCAA

217 484 AAAAATAGCCTTGACTGACAAACTATGTATA CAGGCTCTTGAGACCTCTTTCTC CTTTCTCXAAGGAACTCTGXTCTGGCT TGGGATTGGAATGCAGCAAGCCCAGCAGCCAGATCAGAGTTCCTTGGAGAA

218 486 TTATCTTAGGTTTCGGAGAGCCA CATAATGAGTTCTCAATAAATGTCAGGTC GGCTCCCTCTTCTCTAAAATCCCAA CAGGCCTTGAAAGCCTTCATCATGGGTTGGGATTTTAGAGAAGAGGGAGCC

219 487 AACCAAGTATGGAAGCCTTCTCC CACCCTAGAGGTTAACAGGACCC AGATGTXTCAGGGAGCTTGGAGAAAAG ATGTCTCAGGGAGCTTGGAGAAAAGGTGACAGCCCTGCGGATGAAAGGGTC

220 488 CAACCTCCACTTCCCAGGT CTACTAAAAATACAGAAATTAGCCAGGC TTCAAGXAATTCTCCTGCCTCAGCCTC CAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGAATTACAGGCCCAT

221 489 GGTTTTCAGAGGCATTCAAGC TGCTTCAACAGACCTCAGTATTTCT CAAATTAAATTTAATCACAGGTATT GTCTTTTGTTTTCTGACCAATCTAAAAATACCTGTGATTAAATTTAATTTG

222 490 ACAGCCTCCAGGAGACAGAC CTTAATCACAGCCCTTTGGTATGT CATTCTTTGGTAAGTCCXAGTGTGTCT TTCTTTGGTAAGTCCTAGTGTGTCTAATGAGGTGTCACAGGCCTTGAAGAC

223 491 TTATATCTAGTATGGTAATTTTTGCAGGGT CTAATCTTTACTGTCAGTCTTAGGAAGGTAATTA TCCAGGAAGCAAGCCACTTTAGCTA TCCAGGAAGCAAGCCACTTTAGCTAGAACCCCTGCGGGTTGTCACACAAAG

224 492 CACTTTTATCTTCACAGCGCACT AGTGTCATTTCATTTCCTTGTGTATG TTCXAGGGTTTCTGGGATTCTTTTGGA CCAGGGTTTCTGGGATTCTTTTGGAGAAACTAAAAATACACTAATTTTATA

225 493 AAAATGATCAAGCCTTTCTTTCATG GTTATACTACTACTTTTAGACCAAAGATCCAGC GACCACCAACTCAAAAAACACAAAT ATTAAACAATGTCTGCTTTGTAAGAGATTTGTGTTTTTTGAGTTGGTGGTC

226 494 ATAAGATGAAGTGCAGATGCAATG CTTTCAATTTTTCTCTTCTCTCTTGCTA TTTGTAAAACCACTGTTGCCATAGT AGACCAAGTGGTTGTGGCACTTAGTCACTATGGCAACAGTGGTTTTACAAA

227 495 CATGGTATCAACCTCTATGTGCAT CAAGTGCTGTAAAGGAAAAGCAG GGGAGGGGAAATAGCAAGTGTTATA GGGCAGCAAGCTCATTGATTGCTTGGTATAACACTTGCTATTTCCCCTCCC

228 497 TTGCAGTGAGCCGAGATG CATCATACCTGGTTGGGAGATG ATGGGGAAXCAGATGCAAAATGATGAA AAAAAAAAAAGAACTATTTCTCCTCATTCATCATTTTGCATCTGATTCCCC

229 498 GACAACAATTTGCCATGCATT GGCATTGGCTTAATGACCATT AGTGAAAAATATGAGAGGCAAAAAG TTACATGCATTACAAAAATAGCAATGCTTTTTGCCTCTCATATTTTTCACT

230 500 TATGTACCTAAGTTAAAACTGAAGGGTCC CATCATGCTCTGGGGTCACTA TACAGGACTACCAAXTTCACATCTG CTGTTTACAAATGACACATTTCCATGCAGATGTGAATTTGGTAGTCCTGTA

231 501 TTTAAGTTCTGGGATACATGTGCTG AAACCTAGATGATGGGTTGATGG TGCAGGTTTGTTACATAGGTATACA TGCAGGTTTGTTACATAGGTATACACGTGCCATGGTGGTTTGCTGCACCCA

232 502 CCTAAATACCATTTGAATTGATAAAGATGTA AGTACTAGTGAGTATTTGATTACTAGGTTACACTA CTCAACTCAACTCTGCTTATATGTA CTCAACTCAACTCTGCTTATATGTAATAGATCCATATCCCTTATGTAGTGT

233 503 TAAGATGCAGGTGAAGTGGCTTA AGTTCAGTTCTACTAAAAATCTGGGCA GGCAGGCAGTCTGAGACAAACTCCC AGGAAGTAGTTCAATTTGTATTTGTGGGGAGTTTGTCTCAGACTGCCTGCC

234 504 TGAAGGCAGTTTAGTCACCAAATTA GGGGAGTAGAAATTGAGAATAGGG AGGGGATXGCTAATTTTCTXCAATTCG ACTTGGTTTCAGAGCTCTTGAGTTTCCGAATTGCAGAAAATTAGCAATCCC

235 505 GCAACTTAATCTCCATTGCAGG TCTAGATATTTATGCCAATCCCATCA TTTTATAGCATAGTAXTTTGGAACATT TTATAGCATAGTAATTTGGAACATTGTGTGGCATTTTTATAACAAAAAAAC

236 506 TGTAGGGAAGTATGTTATGCAGAGC AGTCTCTTGTGACAAAAATGTACCAG GACCCAGAAACTGATATXAATGCTTCA CCCAGAAACTGATATGAATGCTTCAAATAAATTCTGCAGAAGCAAAGCTGG

237 507 TAAGATGTCTCTTAATCCAGTTTTCCC AGGACAGAGGGAAGTTGGGA GAGGAGGGTCCAGGAAAGAAGGGAA GTAATCTCTACCAGATAATCCAGCCGTTCCCTTCTTTCCTGGACCCTCCTC

238 508 GGCCTTTCCCACACCAGC CTTCCGAACTTCCCTCTGTCA GAAGGGTAGCCAGGCAGTAGAATTT GAAGGGTAGCCAGGCAGTAGAATTTCATTCAACCCTAGGGTTCCCACCTGA

239 509 GTCAGGCCCCCAGATGAG CTGGTCCAGGTAATAACCCAAAC GGCTGCGGCATCGATTCTGACCACA GGCTGCGGCATCGATTCTGACCACACATTCTGGAAAAATTATCATCCTACA

240 510 CCAGCAGCTCAAAAACACTTG ACCCCTTTTTCTTAGGCATGTTT TGAAAATAAAATAATAAGAAATATA AACATAAATAATAATGTACTTAATAGTATATTTCTTATTATTTTATTTTCA

241 511 CCACTGACGTTATTCAAATGTACAC TCTTTCATAACAAAAGAGAAGAACAGC ACAGATGTGAAAAATAGCATAAGCA ACAGATGTGAAAAATAGCATAAGCAAACACAACCTTAACAAGGCTGTTCTT

242 512 TGCCTCATTATTATGGGGGAA CATGTCTGGACAAACCCTCCT AGGGCACTTGTGTGGTGGAATAGGC AGGGCACTTGTGTGGTGGAATAGGCGGGATGGGGGTGGTGCATTTTATCCA

243 513 ACATCAAATTGTACTGCAATAATATGTACAA TGCATGTTAGAATTTTCTGGGC GXACTGTTGAAAACTCCCCXTGCCCCA TCCACAGAAGTTTTCATTTAATTGTCTGGGGCATGGGGAGTTTTCAACAGT

244 515 TTTCATTCTCAGTAGAGCTTCAAGG TAATGACCACTGGCGCTG CCTGCTGCTCTCTGAACTCCTCTCT TTCCCAGAGAAGGGGCACAGTTCCACAGAGAGGAGTTCAGAGAGCAGCAGG

245 516 TTTATAGGGTTTGGTTTTATTGGGA GAAGCTGCTGACTGCCTGA TGATCTCTGTGAAACTTAGGTGTAA TTTGAGCTGTATAAAACTGTAAACACTTACACCTAAGTTTCACAGAGATCA

Row* REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

246 517 TACACAGACGCTCTTAAGTATCTGACA AAAGATGTCAAGGCTTCAGAAGG GTAGTGACCAGCACTCAAAGATAGC GTAGTGACCAGCACTCAAAGATAGCGCATATGAGCGGCTCAGTCAATCACT

247 518 AAGCAGCGACCCGACCCG GAATGTGTCACCTGCCATGAC TGGGGTCCCTGGGGCAAGCGAATGG TGGGGTCCCTGGGGCAAGCGAATGGCCCTTTGCCTGGAGAACCTGGGCCAG

248 519 GCTAATGCAGTGAGTTTAAGTGTTTAATT CACCAGCAGTTACTAATTTTTTCTAAATTAA AACACTTAAACTCACCGCATTAGCA ATTAGTAACTGCTGGTTGCCTCATAGTGCTAATGCGGTGAGTTTAAGTGTT

249 520 AGAATGGTGTGAACCCGG CCAGAAAGAACTATTTCAGGCTTAGTT TTAGTTTTTTCTTTTTCTTTTTTTT CAACAGAGTGAGACTCCGTTTCAAAAAAAAAAAAGAAAAAGAAAAAACTAA

250 523 TTCAGGCTGCACCCCTTC CTGTGGACTCCCTGACTTCC ACGAACTCGGATGCACCTCTGTGAG ACGAACTCGGATGCACCTCTGTGAGAGGACTGCTCAGTTCACCGGGGTGGG

251 527 TTAAGCAGCTTGCCCAAGG CATGGCGTCGTAGTTGCAG CTAGTAAGCAAGGAACTGAAAATGA CTAGTAAGCAAGGAACTGAAAATGACGCTGGCCTGTCTAACGTGACAGTTC

252 528 ATCAGGAGCACTTGAGCCC GACAAGGCGAGACTCTTGTCTC ACCACATCCTAACATCGTAACAAAA ACCACATCCTAACATCGTAACAAAAGGAGACCCCCATCTCTACAAAACAAA

253 529 CTCCACATTACCAAGAGGATAGGTC GTAAAATGCTCAGCTCAGATGGA AAAGCACCAATAACAACAGTAGCAG AAAGCACCAATAACAACAGTAGCAGCTCATATTCACTAACTGCTCAACATG

254 530 AACATTCTGACTGGCGCTGT CTCATACATCTGTCAGTAATAAAGCCAA GATAAGTAACTGAOACTGAAACGCC CTGTGGTCAGGGAAATGGAATACAGCGGCGTTTCAGTCTCAGTTACTTATC

255 531 TGCTGGGATTACAGGCATG AACCTTTGGCTAAAAGGGTCTG CTGATTACCCAAAGAGATTAGAAAC GCCACCATGCCCAGCCGCCAAGAGGAGTTTCTAATCTCTTTGGGTAATCAG

256 533 TGCTCACCATTTTTAAACATTTATTTG GAGGCAGGAGAATCGCTTG TTCTTTTTGAGGTAGAGTCTTGCTC TTCTTTTTGAGGTAGAGTCTTGCTCCGTCGCCCAGGCTGAAGTGTAGTGGC

257 534 TAGCTGGGATTACAGGCGC GATCACTTGAGGCCAGGAATT AATTTTTGTATTTTTAGTAGAGACG AATTTTTGTATTTTTAGTAGAGACGGGGTTGTGCCATGTTGGCCAGACTGG

258 535 TGAAAACCAAAGCATTGATAGGG GCCAAAAAGGCAACAGGG TGCATTGAGTTGTTGGCAGAATTTC TGCATTGAGTTGTTGGCAGAATTTCGTTCCATGGTGCTGTAGGATTGAGGT

259 539 AGTTTTCCTTTTCTTTTTCCCATG GTAGTAAATGGCATAGAAGACAGGGA GACAGGGAATAAACAAATAATCTGA ATCAATGGTGTGAGCAAGGCCACTCCTCAGATTATTTGTTTATTCCCTGTC

260 543 CACACACACACCCATACATAAAAAA ACGTTTAGATATTTTTCTTTATTGAAGGATG TTCTTTTTATGGCTTGTTTTATATC TAAACATTATAGAAAAAACATTGCAAGATATAAAACAAGCCATAAAAAGAA

261 544 CTGATCTGATTTCACAGTAAGCTNTTTAC TCCCAAAGTGCTGGGATTAC TTGGAATACTTTTAGATATACAGAA TTGGAATACTTTTAGATATACAGAAGAATTATAAAGATAGTATAGGCCAGG

262 545 GTTTAGCCAAAGATTTGAAGCCA CACTCAGATTGTAAAATTTGTAATGCA TAGAAAATCCTGCTTAGTTTAGGCA CAATCCTCTAACGAGTACTGTAGTGATGCCTAAACTAAGCAGGATTTTCTA

263 546 GTACATAAACAGTCCTGCCTGCA AGTATGTTCACTTGATTTACACTCAAGTATTTAG ACTCAAGTATTTAGACCTTTTGTAC TTGGACCCCAGAATAAGAATATTCACGTACAAAAGGTCTAAATACTTGAGT

264 547 CCTTAAAAGTTTTCTTGTTCACGTAATTAT CTTCATTTAAAAAAAAAAGTAGAGGACTG ATTAGTTTGTTAATCGGTGTTTTGG ATTAGTTTGTTAATCGGTGTTTTGGCTGGACCATATGGCCTTTTTTTCTAA

265 548 GGAGAATAGGCTCTCATATGTTAGGTAAG ATGGCTTGCTTCTTCATTTGATT TGATTCCTTTCTCTGCTCCATGTCA AATCAGGAAAGATTTCTTTAATAAACTGACATGGAGCAGAGAAAGGAATCA

266 549 AAGGACCGGACATTTTCACTTC ATATACATCCAGATGGCCTGAAGTAA GTGGAATGTCATCAGTXAAGGCAGGAA GGAATGTCATCAGTTAAGGCAGGAAAAGGCCATTTAAATTTCACTTCTTTT

AGAGACCACAAGAATAACTCTTYGAATATAA TTATCTTTATTATTCATTCXTGTTTAT ATCTTTATTATTCATTCATGTTTATGTATTATTTAATATTAATATTATTAT

268 551 TAGCTTTCAAAAATCTACCCCCC CAAAAGTGGAAAAAACAGATTGACC ACCCCAAATGCTATGCTTTTCTTCA CCCCTCTGCTGGGACATATGATATAGTGAAGAAAAGCATAGCATTTGGGGT

269 553 AAGGAATGTATCCGTCACTCCAC AGGCATTGCTTAGTTACTGGTTGT TGTCTCCGCATATTCAGAAAAYAAT CATGGAAATAAAACCAATCTCAGATAATTRTTTTCTGAATATGCGGAGACA

270 554 GCCAAATCTGTATTCCCAAGTATTT AGAGAGAGAGAGAGAGACTGGTAATAAATAATG ACTGGTAATAAATAATGGXTTAGCAGG AAATGTTCCTTTCTGTTAACTGCAGGCCTGCTAAGCCATTATTTATTACCA

271 555 TTTTAAACATCTGCACGTTTCTGA CATTTTGTAACTCCCACATCTGG TGTTGCCTGCCTCTTTTGCTTCTAC CCAAAAATGTATTTAAATCTCAACTCGTAGAAGCAAAAGAGGCAGGCAACA

272 556 AAGGAGTCCAAAAACCTTTGACC GATCTTGCAGCCTTTTTGCA ACTGAXAGTTGTAAGAACTGACCTTTG TGAAAGTTGTAAGAACTGACCTTTGGCCTAGGATTATGACTCTCATGTAAG

273 557 AATAGCTGACTTCTCTTCCATTTCTG AATGGTACAGCACTTTAGTCATACCAA CATACCAACCCTCCAGAAAACAAAA TGGTATTTTTTTCCAGATTTTATAAGTTTTGTTTTCTGGAGGGTTGGTATG

274 558 TTAGGGACTTAGCTGTCTCTCCCT GATGTTCATAGACATGCAGACAGATT TGATGGCAXTGATATTCAGACACATTA TCRGATTTTTCCAGCAGATATATCTGTAATGTGTCTGAATATCATTGCCAT

275 559 AATCTCTCAATCTCTCAGCCTTTCT CTAACACCGTGTGTGTGTGTGTG GTGTGTGTGTGTGTGTGTGTGTGTG CTCTCTCTCTCTCTCTCTCTCTC CACACACACACACACACACACACACAC

276 560 CTCCTTGTACTTCGAGACCTCAGA TCAGATAAGTAGTACACAAACAATTAAAATAAGG ATTAAAATAAGGAATXCAATAAAGTAA TGGAGGTCTTTTGTGATCACCCAGTCTTACTTTATTGCATTCCTTATTTTA

277 561 GGAGAATCTCTCCCTTCAGGAA GACCATAATGAATTAATCAAAATGGAGA TCTCTTTTTAXAGGCTTTTACCTGATT TCTTTTTAAAGGCTTTTACCTGATTAAGTCAGGCCTTCCTAAGATGATCTC

278 562 CAGCCTGTGTTCCCTGTCC TGACCTTGTCATTTCTTCCATTCT ACCTAGTTTTTTTTTTTGTTTTTTT TAAAAAAAAACAAAAAAAAAAMCCCAAAAAAAACAAAAAAAAAAACTAGGT

279 563 GAATTCTTAATTTACTTTGGCTATCACC TCCAAGAGTCCAGAAGGGG ATTTTGTCATGTTXAAGATCCTCTA ATTTTGTCATGTTTAAGATCCTCTAGTCCTGGATGTGTCTTATACCTCAGG

280 565 GTCATCCCAAGAAGAGATAAGGG TTTATGTAGGGGTTATTTAGGATTTCATAA TTTCATAAACAGATGTAAAXGATTCTT GAATCAGAGAAGAGGAGCCTTCAGACAAGAATCTTTTACATCTGTTTATGA

281 566 TTACTCTGTTGTCTTCTCATTGTTCAAT CAGGAGCTGCCATTTCAGAC CAATAACCACGTCTTCAACTTTTCT GCACTGTGCTAAGTACTGCGAGAAACAGAAAAGTTGAAGACGTGGTTATTG

282 567 AAGTTTACTATGTGAACTTGGGAAAATC TCTGTGCTAAGCACTGGGCT CAAGAGTTTTTTTTATTGTTTCCAC CATATCTTTTCTCTTGTATATTTCACGTGGAAACAATAAAAAAAACTCTTG

283 568 GGGGTCTTATTCTTAGCAGAGCA TGTTGTTTTTGTTAAGAATCTATCATCC GGAAACATAGTAGGCACCCAAGTTA GGAAACATAGTAGGCACCCAAGTTAGGATTTGATGATGCAGAATGGGATGA

284 569 TTTCCCAGGATGGAATTTTGT GACAACAGAAAGAGATGTGGCC CTCCAGCCAGTTGTATAGAGAACAC CTCCAGCCAGTTGTATAGAGAACACGTCAGACAAAACCTTTTCAGATGGAC

285 570 GGACTTGCATAGCAAAAATCAAC TGTCAGTTTTAGTCACTGATAATAACCCT ATGACTAGGAXAACATTTTGTAATT ATGACTAGGAAAACATTTTGTAATTCACTTGTGAATAAAGACTAATTTATA

286 571 AATTCCCCTCTCTAAGCCATAGTT GAATGAACCAGACTTTAGTGTTCAGAC CTAAATGTGTGAGAAAAACAAGCAG CTAAATGTGTGAGAAAAACAAGCAGCCCACACATAGTTCAGGGCCTGGTCT

287 572 GTTTGCTCTGGATCTTCTTTGG TGAAGAAGTCTCCAAATTGTGAAATATAT ATATATTCCTTTGTGCCCAGTCTAA GGTTTTCTACTAAATATTTTAAGTGATTAGACTGGGCACAAAGGAATATAT

288 573 TAATATTGATCTCTGGATTATTGACAGG TGCAATGAAGATTTACTAGATGAAATGA GAAATGAGAGGGTGGGGCTAGAAAA TTAAGTGAGATAACTTGCGTGGTCCGTTTTCTAGCCCCACCCTCTCATTTC

289 574 CACTTTAGAGATAAGGAATTTTACTATACGTTAGA TTCTAAAGAACATTGGGTACTAGATTCCT GAAACCXTAATTATCTATACTAAGAAG AACCTTAATTATCTATACTAAGAAGACCTGATGAAATTATATGTTAAAGCA

290 575 TTATTTTGTATAAGCTATAAAGCACCTAACTATCA TGATCTGCCCACCTCGGC TACXTGTTGGXCGGGCATGGTGGCTCA CATGTTGGCCGGGCATGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAG

291 576 CAGCACCATGCTTCCTGTAC ATAAAGAAATGCCTGAGACTGGG CAGCCTGTAAAAXTGTGAGCCAATTAA GCCTGTAAAATTGTGAGCCAATTAAGCCTCTTTTCTTTATAAATTACCCAG

292 577 TCAGCCTCTCTACTCTAGCCATACTC CAAGAGTTATTCGTTGGTTTGTTTTC AGTCATAATAXAGTCCATXAACCTTTG TCATAATAAACTCCATTAACCTTTGCGTCCAGCTCTGCATGCCCAGTAGAG

293 579 TGCTCGGCTGTGACCTCT CAGTTAGGACTCCATAGCAGGC GGCCTGCAGTGGCCCTGXTGATAACCT CAAGCATTCTTCCATGCTCCTCCCCGAGGTTATCACCAGGGCCACTGCAGG

294 580 TTAAATGGCTTCCCCAATGAC GAAGAGTTCTACTACAATCGTATTGCTGT CCAAATCAATAACAGTGCCCCCAGT CCAAATCAATAACAGTGCCCCCAGTGAAACACAGCACCTTTGTTACTTAAA

Row# REF# Upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

295 581 GCATTGCAGAAACAAAGTTCAGT TTAGGCCATTGCTYTTTTGATG TTGATGTTXCAGAAACAGTGGATTCAG CTTTTAAATCCTACTGGGCAACCAGACTGAATCCACTGTTTCTGGAACATC

296 582 CATGCGAGTGGGACCATG CTCTTCCTCCTCCTGGTGTCTT ACTCCCCACTTCACACTCCCATCTA GACTGGAAGTGGGCCATCCAGACTAATAGATGGGAGTGTGAAGTGGGGAGT

297 583 TGTACAGCAGTGCCAGAGAAAA CACATCAACCTGTATGATCTGCC TTGAGATAATCCTCCAAAACTACTT TGTGTCTCAAAAACCTTCAATTAAAAAAGTAGTTTTGGAGGATTATCTCAA

298 584 CGCTACTTATTTATTTCTATCCCAAGC CGCCCAGCAGCAGCTTCC TTTGCCCCTCCCAACTTCCASCCTG CAGCAGCGGCGATCTGCGACTGCGCGCAGGΞTGGAAGTTGGGAGGGGCAAA

299 585 CGAGAGAGTATATCAAGAAGGCAAAG TATGCTCAGAGTTTTGTGAGTCAATT ATATTTACCCTGCAATGGAAAATTT ATATTTACCCTGCAATGGAAAATTTCGTCAATTAGGCAAATTGAATTGACT

300 586 ATCACGAGGTCAGGAGTTTGAG GATTACAGGCAMCTGCCACC GGCXAACTTTTGTATTTTTAGTAGAGA AGCCTGGCCAAGATGGTGAAACCCCGTCTCTACTAAAAATACAAAAGTTAG

301 587 AGATGGTGAAACCCCRTCTCT GATTCTCCTGCCTCAGCTTCC TCCTRAGTAGCTGGGATTACAGGCA AAAAGTTAGCCGGGCGTGGTGGCAGGTGCCTGTAATCCCAGCTACTYAGGA

302 589 TCACATCGGAGACAGACCTATAATC CAATGTAATTCACTGAAAGYATTCCTAG CAGAGCTTGGACTAAXGACTTGCCCTA CAGGCTCCCATATGTGAATACCTAACTAGGGCAAGTCCTTAGTCCAAGCTC

303 590 TGCTGTCGTGGTCTTCCTC CATTTCTTACCAATTTCCCCAAT ATAGGATTACTCACTCATTCACGCA ATAGGATTACTCACTCATTCACGCACTCATTCATTGGCCAGATCTGTAGTT

304 591 CAACACCTTTATCCTTTTCTTCGAT TCAATTCATGCCAAAAATATCATG TCATGTAAGATGAAAGTGAGGTATA AAGGTCCTGGGGATACCTTAACCTCGTATACCTCACTTTCATCTTACATGA

305 592 TCACTGCAACCTCTGCCTC CAAAAACTAGCCASGTGTGGTG CAATTCTCTTGCCTCAGTCTCCCAA CAATTCTCTTGCCTCAGTCTCCCAAGTAGCTGGGATCACAGGCACCCGTCA

306 593 GGGTTCAAGCAATTCTCTTGC CATGGCAAAACCCCGTCT GGATCACAGGCACCCGTCACCACAC GGATCACAGGCACCCGTCACCACACGTGGCTAGTTTTTGGATTTTTAGTAG

307 594 CGATCCTTTACCACATCCTTCTT TGTGAGATGATGGCTATGTTCATT TTGTTCCACTGTAGXAATCATTTTACC AACATGATGTTTTAAGACACATATACGGTAAAATGATTACTACAGTGGAAC

308 595 CTTGGAAAGCAGGTATATGGAAGG TCTGTGTGTGTGTGCCTGTGT TCATTTCACTAATGTTGGCTCCTCT GTTATCTCAACTTCACATTAAACATAAGAGGAGCCAACATTAGTGAAATGA

309 596 TAGATACTAATAACTTCTATTCTCTGATGCATTG GATTGAGGCTACAGTGAGCTGTG TAGTAGGGCAGACATXAAGCATTTCAT GTAGGGCAGACATTAAGCATTTCATGTGCATTTTTTTTTTCTTTTTGAGAC

310 598 TTCCCAAACTACTTACCGGAGC CTTGGCCTCCCAAAGTGTT GATTTTTTAATGTCTTAGATGGAGC ACAATAGGAAGAACCAGGGATTGCACGCTCCATCTAAGACATTAAAAAATC

311 599 GCCCCTGTAGTCCCAGCTA TTATTTTTTTATTGAGACAGAGTCGCT TTGGGAGGCTGAGGCAGGAGAATGG TTGGGAGGCTGAGGCAGGAGAATGGGGTGAACCCCGGAGGCGGAGCTTGCA

312 600 CATGCATAACTCTGACAGGAAGA GGGACACCCAAAAGCTGAA AAGCACAGAGGGGTTGGCGAGGAGT GCCCCTGGGGAGGGAGAGGTCCAGGAACTCCTCGCCAACCCCTCTGTGCTT

313 601 GTGTTTAATACCGTTTCTTCATCTATAACATAA TGCCACAGAGAGACTTCTTTTTTT TAATGATATAGGAAGCCACTCAAAT TAATGATATAGGAAGCCACTCAAATGAGGCAGACATGTTGCGTTGCGCTTA

314 602 TAATGGGTGTCTCCTTATGATATCCA CTTTTTATACTCCACTCTGCAAGCG TATGCAGAGTTCTGGAAGAATTCCC TTTGATGGCCTTGCTAGCTCAGTGAGGGGAATTCTTCCAGAACTCTGCATA

315 603 TCGGCCTTCCAGAGTGCT GAAATTTTATCATGAGCCAGATGC TCATCATTGAAATTAGAXCTTCTGGCT GATTACAGGCGTGAGCCACCGCGCCAAGCCAGAAGCTCTAATTTCAATGAT

316 604 CTTGCAGTTTCTTCCTTTGTTCTACT GAACACAATGATGCCAAAAACC GTTGTCAAGTTTAATCCTTCCTCCT GTTGTCAAGTTTAATCCTTCCTCCTGCAAACTCCCTGAGGCAGACTCTTTT

317 605 ATTACAGGCGTGAGCCACC GTGTTTCTCCAGGACCGAATC CATGAGCCAGATGCTGAGGAAGAGA AATTTCAATGATGATTGTGCTTTTTATCTCTTCCTCAGCATCTGGCTCATG

318 606 TAGTGTGGATTCTCAAGTTTTCTCCT CTTGTTAGCTGCCTTCCTTTACC ACTTCCATCATACAGTCTTCAGCAA ACTTCCATCATACAGTCTTCAGCAACCTTGAAAGATTGGACAAGCTTCAGC

319 607 AAGTAATCCTCCTGCCTGAGACTC ATTTCTTGAGCCCAGGAGATTAAG ACGAGACCCCATCTCTACAAAAAAA CTACCACACCCAGCTATTTAAAAAAATTTTTTTGTAGAGATGGGGTCTCGT

320 608 GCAGCATTTTTCATAAGAGCTAAACA TAGCATAATGTTTTAGCACACATTGG CACATTGGTAGTTTATTTCTTCTTA ACATGGAATAGAATATCACTTAACTGTAAGAAGAAATAAACTACCAATGTG

321 609 CGTGTACCGAGAGCTGTTTT CAGTCCTCTTGGTCTGGAGAGG GAGAGGATGGGXCTGGXCTGGTGCCAC TGCTAGATCCCTCAGCACCTCTGTGGGTGGCACCAGCCCAGCCCCATCCTC

322 610 CAGGAAGGTGGGGAAAAGTG CAGACAGGAAGCAGGGGC CGACTGGGCATTGGCAAGTGAAGGG CGACTGGGCATTGGCAAGTGAAGGGCCCTGGAGGCGGGGAGGGGGCATCTC

323 611 TTTTTTTATATTTGGAGTACTGCTCAACA ACATGGCTCAAGATTCTATCATACAAC TATCATACAACATCTXCTCGCATTCAA ACTGACCTTGGCGTGACTTTGGCCAGTTGAATGCGAGCAGATGTTGTATGA

324 614 AAATGTGTATCAAACCACAATCCTCT GTTTCTGGCAGAATGTTCCAAA ATAGGAACTAGCAGCCAAAGGACAA CACTCCTGACTCGGTCATTTCTGACGTTGTCCTTTGGCTGCTAGTTCCTAT

325 615 TAGGGAACCACTTACTACTCATAGAATCAT GTTTCTATCTTCATTTGTTTCTATACCCTTT CAATATGTAAGATGGAXAAGATTCTAT AAGAATTTGCCTGGAAGCAATCTCAGATAGAATCTTCTCCATCTTACATAT

GGAGAACTGCTTGAACCCAGA CCAGAAGXCAGAGGTTXCAGTGAGCCA CAGGCTGGAGTGCAGTAGTGCAATCCTGGCTCACTGCAACCTCTGCCTTCT

327 617 TTGCACATTTTTCCCAATGAA ACTTGAGAGCAGCGTGATTCC TGAAGAAGGCATCTCTGGATAATGA TCATCAGGACGCTATTTCTTATGCTATCATTATCCAGAGATGCCTTCTTCA

328 618 GAAGTCAGGGTGAGGGGACA CGCCATTGCACTCCACCC _{CATTTTTTTTTTTTTTTTTTTTTTTGTGAGATGGAGTTTCACTTTTGTTGC}

329 619 TTGCTTTATCATCCCTCATCA GTTTTAGAACAGGGCTAAAATGGTC GAGAGAXGGTGACAXGGAGCCCAGGGA ACCCTTTGTCATAGCTGCCCCCTTCGTCCCTGGGCTCCCTGTCACCCTCTC

330 620 ACTAAAAATACAAACTTGAAAACATCATGG CAAAGTCCTGGGATTACAGGC GGCTGGCTCAATTCTTTTGGCACAA GAAGAAGCCTCTGGGATCAAAAATAGTTGTGCCAAAAGAATTGAGCCAGCC

331 621 TATAATGGAGCAAGTCTTACTTTGTGAG GAAACACTGGTCCCATTGGAT TATTCAAAAGCTAAATGTGGGTCCT TATTCAAAAGCTAAATGTGGGTCCTGTGAAATGCTTTCCCATTCAAAAGCT

332 622 CTGTTAGGAAAAAGTTCACTCCTCTCT ACCTTAATTCTCTCCTTAAAGGCCC GGCCCTATCTCCAAATGTAGTCACA TTATATGTTGAAAGTCTAGCTTCCAGTGTGACTACATTTGGAGATAGGGCC

333 623 TTTCCTTTCATTTCTTTTCTTTGAGAT GAACTTGTAAGGTGCAGGTTGC TTGCXGTCAGCTGAGAATGAGCCACTG CGTCTCAGTCTTCCTAAGCTGGAGCGCAGTGGCTCATTCTCAGCTGACTGC

334 624 TGCAGAGCCCATCTCCCC GGCCTCCCAGAGCCTCAC CTTXCCTGCAGCAGXCTGGTAGGGCAC TCCCTGCAGCAGCCTGGTAGGGCACAGGCATTGCCACTCTCTAAGAGGACA

335 625 TAAAAAAGATGCTTCACATACACCCT GATTTTCTCCCCTACAGGACACC CAAGTGCAGGCAXACAGTGTGACAACC AGTGCAGGCACACAGTGTGACAACCGAAAATGTCTCTGACATTGTTAGGTG

336 6₂6 ACATAGTAAATGCTCAGTAAATGTAGGCA CTCTTAATAGCAAAAATAAAATATGAGCTATAGG GACCAATAAGGCAAGAGXCCAGACATA TTAAGAGGCAGACGGAGGGCCTGCAGTATGTCTGGCCTCTTGCCTTATTGG

337 627 AGATCTCAGGAGGAGGGCTG GTACCTTTCAAATGCTGGCC GGXCCAGAAAGGTGAGGCAAGGAGGGG GGGATTCACAGAGATGGAGGCCAGGGCCCCTCCTTGCCTCACCTTTCTGGG

338 628 AAGAGAAATAACTAGTCTTCCTGTCTCAGG GAACGAGAGATAGAAAAACTGAATAGTAGTTG TGGGAAAAATATCACATATAAGTGG TGGGAAAAATATCACATATAAGTGGATACATGCAGCTCAATCCCACGTTGT

339 629 ACCAACTAGTGCCAAGCGC GAATTCTGGAGACACGCTGC ACAGCTATGACXCTGCCTTCTGAGAGG AGCTATGACTCTGCCTTCTGAGAGGCTTTCTGGAAAGAATGAGTTCTCTGG

340 630 TTGGGTATTTGTTTATAGCAACACAA GAGGTTCCCCAGCCTTGT TCCTCCACTCCTCCTTCTTTCCCCC ACTAACATGGGGGTGAGGTTGGAGCGGGGGGAAAGAAGGAGGAGTGGAGGA

341 631 CAAGCACCTGTCCACGGT CTTATACTTTCCTTTCTACTGCGGG CTGXCCAGAGCACAXAACGGGGTTCCA GCCCAGAGCACAGAACGGGGTTCCAGAGTGCACTCACCTCCCCTCTGAACT

342 632 TCTAGCCTTAGGGTACCTTCCAAG GATATCAAACACAGGACTTAACCCA TGTGACCXAGATATATTTGTGGCTTCT TGACCCAGATATATTTGTGGCTTCTCGTCATATTTGCCTTTCCAAATTCAG

343 633 TTTTCTGGACTAAACTGCTGCG CAGACTCAGAGAGATTAAGTGACTGGA GTAAACAGTAGAGCCAGAGCCATAA ACATGTAACAGAAAGAACAGTTATAATTATGGCTCTGGCTCTACTGTTTAC

RθW# REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

344 634 AGGACTAACCCCACATTGGG CTTGACTAGTTCCCAGTCGAAATTT CCCCAGTCATGGGGTGATTGTCCTT CCCCAGTCATGGGGTGATTGTCCTTGTTGAGCCCAAGGAATGATGGTGTTT

345 635 AACAGACAGACCCCCACCC CAACCCCTTTCTGATTTCCC CACAGCCACTCGGCTTCCTTCACTC GCAGCCTGCTCAGGGAGCCAGGGCAGGAGTGAAGGAAGCCGAGTGGCTGTG

346 636 TGCAGACTCCCGCCCAGG GCCAGTTGTTGAACGTGGG GGGGTTGTGTGACTCAGAXCCTGGGCT CCACACTGCCAGCTGGGATTGGACAGAGCCCAGGCTCTGAGTCACACAACC

347 637 CAACAGACATTGCATTGGCA TGGCTTAGAACTCAGAAACCAGG TGACTGTAGTTAAATAATCTCCTAT TTGCTTTTGGTTTTCAGCTGTAATAGATAGGAGATTATTTAACTACAGTCA

348 638 TGACCCCTACAGACACTTAGTTGTG GTGATGGTGCTGACGTAGGATT ATTTXGGACTATCAATCCCTATGTC AGAAGGCAGTTCTCTAGAGTGGAAAGGACATAGGGATTGATAGTCCTAAAT

349 639 GCCTCCACTTTTGGACTGACT AGAGCTTGCTTTAAAAATGCCAC ATGTGAGAAGAXTTCCTAAXTTACCAC GTGAGAAGAATTCCTAAATTACCACGTATGAAATTAACGTCCATCTTTCTG

350 641 TTTAAAGTGGCCCTGCTGTTAC GATTACAGGCATGAGCCACC CCAGCGGXTTTAGTCTCAGGTAAACCA AGCGGTTTTAGTCTCAGGTAAACCAGTCTTGGTCATGATTCTCTGTCTTCA

351 643 TTAGGTGTTACAAGTGATCTGGAGATG TCTATGGATGCTCAAGTCTCTGATATG TTAAAGTATGTGGGAGAATGCGGGT TTAAAGTATGTGGGAGAATGCGGGTGGTTTATATGCAAATACTGTGAAGGC

352 644 TTTTTTTTTTTTTGAGACAGAGCAGT GCAGGAGAATCACTTGAACCC AXCCCGGAGGCAGAGGTTXCAGTGAGC CTCTGTCACCCAGGCTGGTGATCTCGGCTCACTGCAACCTCTGCCTCCGGG

353 645 ATCCCCCTCTCTGTCACCC GGTACCAGGGAAGATGAGGG GCCCAGGAGGTGGAGGTTGTAGTGA CTGGAGTGCAGTGGCATGATCACGGGTCACTACAACCTCCACCTCCTGGGC

354 646 TTGGGGGGAGGCAGCCAT CTCAATGGAAAGTCCCGTAATG AGGATTAAGTGAGATGGTGCCTGGT GAGCACCTGCTCCTCACCAGGCATTGACCAGGCACCATCTCACTTAATCCT

355 647 GAACCCTGCGAATACACCCT CTCATCCTCCCACCTTGGC TTAAAATGCTACCGTATAATOTATG GAGTCTGAAATTTATTCACACATATACATACATTATACGGTAGCATTTTAA

356 648 CCCACTGGAAGGTGAGCC CAATTAACCTGGACACCGCC CAAGAGACTGGGCCAACACCGAGCA CAAGAGACTGGGCCAACACCGAGCACGGAGGGGATCAGAGCGTGTGTGTGT

357 650 AGTCTCTCCGCTTGGGTCTT GGGTTCTGAGCCCATGATATC CACTGAGCACGTGATTCCCCACACC CACTGAGCACGTGATTCCCCACACCGAGCGAACTTCAAAGCTCCCAGTGTA

358 651 TTGTGTGTGGAGACACTGAGC GCTGAAAGGGAGGTGTGATTT ACACAGCCACGATACAATGTCAGCC CCCACAACTGTCTCCTGCAGCATGAGGGCTGACATTGTATCGTGGCTGTGT

359 652 TGCTCCAGAATCCAAAACATTT TGAGCATCCCCAATCCAA TCTGAAATTTAGACTGCTCCAATGA ACCAAAATGATGTTCAAAGGAAGTGCTCATTGGAGCAGTCTAAATTTCAGA

360 653 GTCCAAAATTAATCTAACTGATCCAACTT AGGACCTAACTACGTGTTGTCTACAAG ACAAGCAATGCACTTTGAATATAAT CCCTCTACTTTTCATCAAGCTGACTCATTATATTCAAAGTGCATTGCTTGT

361 936 CTCATGAACCAACAGTATAGAAGAAATAATT TCACTGTCAGTGGCTCTAATTAAGAAT TGGTGCGATXTGGGCAAACXAGAAAAA GTGCGATTTGGGCAAACTAGAAAAAGMCAAGAAAAATGAAAAGATCTTTTT

362 937 TGATTTTCCATACAAGCTCACATATAC AAATATAACTGAGATTGGCTAACAGCTT TTAAGACCGAATTCTCTCXAGGCAAAC CCATGAATCCGAGACTCGAACAGATCGTTTGCCTAGAGAGAATTCGGTCTT

363 938 TCTAGTGCTGAAATTTTGGTGTGA CAATAGCATCCTATGATCCAGCC GAATTCCCTAGGCAGAAAAAAAGGA GAATTCCCTAGGCAGAAAAAAAGGAGAGGTAGAAGGAACAGCATATTCTAA

364 939 TCTGAAATTTTGGAACAYACTGG GTATGTGCTTAATTCCTCCAGCA GTCTCTAATGAGTTTTTTCTGGTAG GTCTCTAATGAGTTTTTTCTGGTAGGCAATGTTTTACATGTGTTGTCATAG

365 940 GCAAACAAGCCTGTGGTATAAATT TCCCAATCCAGTAGACGCAG AACTGTGCATATAAGAXTTXAATGAAG TTTATCCACAGAAAGCAGACTGCCACCTTCATTAAATTCTTATATGCACAG

366 941 TGAAAACCAAAGCATTGATAGGG GCCAAAAAGGCAACAGGG TGCATTGAGTTGTTGGCAGAATTTC TGCATTGAGTTGTTGGCAGAATTTCGTTCCATGGTGCTGTAGGATTGAGGT

367 942 TAGCTGGGATTACAGGCGC GATCACTTGAGGCCAGGAATT AATTTTTGTATTTTTAGTAGAGACG AATTTTTGTATTTTTAGTAGAGACGGGGTTGTGCCATGTTGGCCAGACTGG

368 943 ATTACAGGAGTGATTCCAGGTGC GTCACACAGTCATGGAGACATAATACC TCTGTGGGAACAGAGAGAGCATCAC TCTGTGGGAACAGAGAGAGCATCACCGTAGTTCAAGCCAGGCTCCTTTAGG

369 944 TGCTGGGATTACAGGCATG AACCTTTGGCTAAAAGGGTCTG CTGATTACCCAAAGAGATTAGAAAC GCCACCATGCCCAGCCGCCAAGAGGAGTTTCTAATCTCTTTGGGTAATCAG

370 945 CTCCACATTACCAAGAGGATAGGTC GTAAAATGCTCAGCTCAGATGGA AAAGCACCAATAACAACAGTAGCAG AAAGCACCAATAACAACAGTAGCAGCTCATATTCACTAACTGCTCAACATG

371 946 ATCAGGAGCACTTGAGCCC GACAAGGCGAGACTCTTGTCTC ACCACATCCTAACATCGTAACAAAA ACCACATCCTAACATCGTAACAAAAGGAGACCCCCATCTCTACAAAACAAA

372 947 CTGCAATTCCTGCCAAAAGT TGGCTACTGTTGCTTTACACTGAA AAAGATTATTGGAAAAXTTGACAAG AAAGATTATTGGAAAAATTGACAAGATGATTTAAAATTATATAGAAATACA

373 948 ATATGACAGCTTGACATGATTACGAA CTTTAGGATAGTAGGGCCTGCTC GTGAGXGCTGGTGCAGACAGGACCC GCTTATTCCAGATAGAAGCCGCCAGCGGGTCCTGTCTGCACCAGCCCTCAC

374 949 TCCATGTTGACCAGGCTAGT AGTAAAGAGAGAGAACACAAAAGAAATTTTT TCTTGAACTTGTGAXCTCAGGTGATCT TTGAACTTGTGACCTCAGGTGATCTGCCCGCCTCGGCCTCCCAAACTGCTG

375 950 AGGAGCTGAGGGATCTGGG TAGAGAAGATGCAGAGAAGAGGAGAC AAGAGGAGACCCXTGCAAAGTCATGCT GTGTCCCATCTTGGCCACTATTCACAAGCATGACTTTGCATGGGTCTCCTC

376 951 AAAAGGAGATCGCAGTTCGA GCGTGGAATCTTGATTCGTG TGAGAAGGAGGGAAGAGGTTGTTTC CCGATTCCCTCTCCCCGCCGACTCTGGAAACAACCTCTTCCCTCCTTCTCA

377 952 ATGCACACACCTTGCATGG AATAGTCAATCTGCATCCATGTCC TCACAAGGGGGCTCCACCAGACTCA TCACAAGGGGGCTCCACCAGACTCACCACCGAAGAAATGCCAGCCAACAGC

378 953 CAGGTGATCAGGAAGGACACTAG GTGTTTTAGGTCATATTTCTTGGGG GTGGATTTATATGAAAAAGGAAAAC GTGGATTTATATGAAAAAGGAAAACGACATGCAGGCTGTCTGTACTAAGCC

379 95₄ GCCTTTCTGCACCTAATTCATG TGTTCAAAATATATACTGAGGTCACTACTGA CCCAAGATTTCGTCACTCCATAGTA CCCAAGATTTCCTCACTCCATAGTAGAGTAGTTTTAAATTTTCAGTAGTGA

380 955 GTTCTATATAGGCAAACTCATTTGGACA ACGGTTACCATCCCAAAGTCTAA TACCATCCCAAAGTCTAAGAAAAAT CAATTTTATCTTTGAATGTTCCTTTCATTTTTCTTAGACTTTGGGATGGTA

381 956 TCTCccTTTTTTTTTAACTTCcc GCCAATGGTTGCCAGGTT ACGGAGXCTGCTGGCTTTCAAGTCTAC TTTCATTGTCCCTCCACTGATCCACGGTAGACTTGAAAGCCAGCAGTCTCC

382 957 CTTAGTAAGCAACACAAAAACATTGGT GCGCCACTGCACTCCAGC AAGTAXACXAAATAGTATGAAATAAAA GTATACTAAATAGTATGAAATAAAAGTCTGTTTAAATTTTTTTCTTTTTTT

383 958 AGGGTCACTTGAACCCTGGA GATCCATTTTTAATTGAGAGATTTCTG CTGTTCTCTAGCCTGGGTGACAAGA CTGTTCTCTAGCCTGGGTGACAAGAGCGAAACTCCATCTGAAAAAAACAAA

384 959 ATAATAGAACATCTAAAGTCCAAGAAGAGT TCTTCCTCCAATTCCCTCTGTAT TCTGTATGCTXAACTGTTTTTGAATGC TGGGCGAGGTTCATTGGGAAATTAAGGCATTCAAAAACAGTTGAGCATACA

385 960 GTACCCATGAGATCTTTCACCTACAC TGTCTTAAATCCTTTCACTATAAGGACC ACCCTTXAAAGTTGCCTAAGTTGAAAT ATCTAATAACTGAATAATGGCAATTCATTTCAACTTAGGCAACTTTGAAGG

386 961 GAAAAACATTTCCACTGAAAACCA ACCGGGGATCTCTGGGAT ACCGGGGXTCTCTGGGXTCAGGTGAGT TGGGAGAACTTCAGCTTTTTTTCCTCACTCACCTGATCCCAGAGATCCCCG

387 963 GCCTTTCCTTAACTAGCCTGTAATCT ACGATTAGCCAGACTGAATGCA GTAAGTACATCTTACATTTTTATTA GTAAGTACATCTTACATTTTTATTACCTTTAGCATCTATTAGCACAATGCA

388 964 ATCTAAGGCTCCAGGGTCTCC CTTGTGAACCCCCTCACCA CAGCTCTGTGTXAGACTGAAGACCCAA GCTCTGTGTCAGACTGAAGACCCAAGGCCCTGGTGAGGGGGTTCACAAG

389 965 CTGCTTTAAACAGTGGCTGAACTAA TGGGAGGCTGTGGAGAAA GGCTGTGGAGAAAAGGGAACATTTA GCACCAATTATGTTTAAATGTTCCCGTAAATGTTCCCTTTTCTCCACAGCC

390 966 GTACTCAGGTTAGGTGTGGTTCAC ACTTACCCAAGTCCATATCTTAGGTTATCT TTAGXTTXTCTTTATAACCCTACACAC TCACTTTTGCTTTAAGGGAATTCCTCGTGTGTAGGGTTATAAAGATAACCT

391 967 GAGCTTTAGAGGAACTAGACTTTCTATCCT AGGCCATCACCATCTAAACCTT GTGTTGATTCTTCTTTTTATCCCAA GTGTTGATTCTTCTTTTTATCCCAACACTTAACGTGTGAATACATCCTGAC

392 968 ATTAAAGCAGTTTCCTAGCGTGTG CAGCCAAACCTGCCTTCC TCTGCCAGCCAAACCTGCCTTCCTC AATGCTTTCACATGAAGGGATATTTCGAGGAAGGCAGGTTTGGCTG

RθW# REF# Upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

393 970 CATCCCCCATAGTCCTGCTC GAAAATGTGTTGGGAAAGGCA GCTTCTTAGGCAAACATTTCTTTTT GCTTCTTAGGCAAACATTTCTTTTTGGTTTGAGGCTTTTATAGCTGCAGGG

394 971 TGGAAAATATGGGAAAATGGTAATG GGATCTTTCTGGYGCCCA AACAAGTGGTTGAGCAGGGGAGGGG AACAAGTGGTTGAGCAGGGGAGGGGGGTCATATCTGGGCRCCAGAAAGATC

395 973 TGCCTTGAAGTGTTTTTGCTG CTCAAATAAGAGCTGTGTGGCC CAGAGAGGTCCAGCCACTTGCCCAA CAGAGAGGTCCAGCCACTTGCCCAAGGCCACACAGCTCTTATTTGAG

396 975 GGCCTAAGCCACTTGTCCTG ATAGCTGCCTCTTCCCTTTTATAAG CTGCATACTACATAGGTAAAAGATG CTGCATACTACATAGGTAAAAGATGGGGACAAATACTTTCTATTTGCTTAT

397 977 GTGAGTAAATACAGAAACAGATGGATATTTT ATGTCCATTACAGATTAGCCCACA AGCCCXCACTTAAGTTATTGCTGTGAC ACGGACAACAACAGTGAAAAGGATCGGTCACAGCAATAACTTAAGTGTGGG

398 978 ATTTCCTTCCTAGAAGGTCCCAG GATATTTAAGCAAATACTGTAGGATAACAGCT ATAACXGCTGAAGAGGCTGGAGAGAGG CTGTGATTCTCTTTCCTATCATGTCGCCTCTCTCCAGCCTCTTCAGCTGTT

399 979 CAAAGGAATGTATCCCTCACTCC TGCTTAGTTACTGGTTGTCCTGAAA AATTGCACCCCTAGAAAACCTATTT AATTGCACCCCTAGAAAACCTATTTCATGGCATAATTTTTGTTCTATTACT

400 980 GTGTGGCTAGGAGAGAGATGCA TGTCCAGGGGAAAATATGTTCTG AAGAATATTAGGAAAGCAAGTTGAA AAGAATATTAGGAAAGCAAGTTGAAAACTTCAGGAAGAAGAATGAAAATCA

401 981 ACTCGTAGCACCTTCAATGGTATAAT CAGGAGGAATGAGGACCTTTAAG AGACTTTCACACTGTTCTCTATTGA AGACTTTCACACTGTTCTCTATTGACGTTCTCTTCTATAGTGTTGATAATA

402 982 ACCAGTAGCCAGCAATTCTGC CAATAACAAACAGTGAGAGTCATCCT TGCCAGXGCTGTTCGCCTATTCAACCC CTGCCTCTAGAATAAATGGTTACCACGGGTTGAATAGGCGAACAGCCCTGG

403 983 GCAGAATTCACAAATATATAACCTCATAATT TCATGCTTAAACATCAACCTTGTG TGTGAATARAGTAGTAXGAAATCTTGA ATATATAACCTCATAATTCATCGACGTCAAGATTTCTTACTACTYTATTCA

404 984 CAGAATTAGAACTGGTATATGAAGAGAGTAGC GGATCAAGGACAACTCCAGGA AATAXTAXTATXAAATTATTGAATGAT GTATATGAAGAGAGTAGCCCATGGAAATCATTCAATAATTTGATATTATTA

405 985 TATATAAGCATACGTTGGCCCAAT CATTTGAATATGTCAGCCTGAGG GATATATAAGCATACGTTGXCCCAATG TATATAAGCATACGTTGGCCCAATGGTATTAATTTGCTTATCCATACTAAT

406 986 AAGGTTTATTTCAGGCCCATTATAG GTGTGGTGCTGCAACAGC TTATTTCAGGCCCATTATAXCTTTCCA ATTTCAGGCCCATTATAGCTTTCCAACTGATCTCCTGCATTCCTTTTGGTT

407 987 TGCAGTGTCATTTGGTTCACTT GATACTATTACAAGTTGTTAGGAAACAGTAAATTA GGATCTGCAXTGTCATTTGGTTCACTT GTACCCGGGGATCTGCAGTGTCATTTGGTTCACTTCGCTACTACTGAA

408 988 CCTGACCGAATCCCTATCTTCT AGGAGAAAACGTATGTAAAGGATTTAGTAGA TCTTAGAXTTGAGAAATAATAATTG TCTTAGAATTGAGAAATAATAATTGCTTAATTTTAATAAGTTTCTACTAAA

409 989 CTGGACTGGATGGCCTCTG CACACCTATTTCCTGTAGGTTTTAATTATTT TTTGGGGCAAAGCTGGAAAATGAAA TTTGGGGCAAAGCTGGAAAATGAAAGGATTTCATTTGGCAAATAATTAAAA

410 990 ATTCCACCTGAATGCACACTG GATCTCCTGGAACAGAATGCTATG CAAGACCTCAGTATCTGCTTCAGTG CAAGACCTCAGTATCTGCTTCAGTGGTGTAACTGTGCACCTGCAGCCATAG

411 991 AGCGGCAACCTGGTCCCA CTTTCAGAGGCAGATCTCGG GCGGTGAGGCAGGAAATGGGTTGTT GCGGTGAGGCAGGAAATGGGTTGTTGAACAGGGAGAAGGTCAGGAGCCGTG

412 992 AACAGAGTGAGACTCCGTGTCAG CACACTCAGGGGGCTTCA AAAGACAAAACTAAGCCTGAAATAG AAAGACAAAACTAAGCCTGAAATAGCTCTTTCTGGGCCTCCCCTTTGCTGT

413 994 ATCCCCCTCTCTGTCACCC GGTACCAGGGAAGATGAGGG GCCCAGGAGGTGGAGGTTGTAGTGA CTGGAGTGCAGTGGCATGATCACGGGTCACTACAACCTCCACCTCCTGGGC

414 995 TTGGGGGGAGGCAGCCAT CTCAATGGAAAGTCCCGTAATG AGGATTAAGTGAGATGGTGCCTGGT GAGCACCTGCTCCTCACCAGGCATTGACCAGGCACCATCTCACTTAATCCT

415 996 CCCACTGGAAGGTGAGCC CAATTAACCTGGACACCGCC CAAGAGACTGGGCCAACACCGAGCA CAAGAGACTGGGCCAACACCGAGCACGGAGGGGATCAGAGCGTGTGTGTGT

416 997 GCAGGAGACTCGCTTGAAC GTTTCACGCTTGTTGCCC CCTGCGAGXCGGAGGTTACAGTGAGCC TGCGAGGCGGAGGTTACAGTGAGCCGAGATCGTGCCATTGCACTCTAGTCT

417 998 CTGGTGAAAATTAAGTTAGCAAATGAA CCTTTATTAGAAAGGAGAGTTAAATGTTGTATAC CACTTCCATATGGTTTCTTTTCTCC CACTTCCATATGGTTTCTTTTCTCCGACATGGGACTATTCAGTTGACCTTT

418 1000 CGTCCTTAATTTGTTCTTTGTAGCA ACTGAAGCCTGCCAATGAAC CATGCAATACAXATCATTTACTAATAT TGGTGCAGTAGAATAGACCAACCTCAATATTAGTAAATGATTTGTATTGCA

419 1001 TCCCCAGACATGTGGAATTG CATTTTTAGATGTCTTTACAGCAATGC GCTGATAAAGACATAACCXAGACTTGG ACTAAACCTCTTCCCTTTATAAATTACCAAGTCTTGGTTATGTCTTTATCA

420 1002 AAAAATGCTGGGTGAAAGAAGC CAATCGGGACTCCTACCAAATT ATTCCCATCCAGXTTTACCAACAGGCA GAAGTAGAAGATGGATCATTGGAATGTGCCTGTTGGTAAACCTGGATGGGA

421 1003 CCAGGAGTAGGGCATTGCTAT CCTCATCTTTCTGTCTTTTTCTAAGC AAACTGTTCAAATCTCTGCCCATTA AAGATACCTGACACTTTAAAACTGGGTAATGGGCAGAGATTTGAACAGTTT

422 1004 GAATCACCTGTCTGGGTCTCAA GATAATTCAGAGAAGGATTGTCCACA ATTGTCCACXTTAAAATGXTCCCTTAC AATGGAAGTGCTTTAGCTTCACTCAGGTAAGGGATCATTTTAATGTGGACA

423 1005 GCATTTCTGAGTCCTCCTCAAG AATTTTCAATGAGATACTGGCTTCTATTT GGCTTCTATTTTGCTTATTGGTTTG AGAGTCAGGCAATCCTCAGTCATGTACAAACCAATAAGCAAAATAGAAGCC

424 1006 TTAAAATAACTACAGTCATTCCAGTGGAC TTTAGATAAAATCCAAAACTCTTCCTCTC CAGATGCAAGTYGGAGAACCXTCAGGC GATGCAAGTYGGAGAACCCTCAGGCGGAAGCTGAGCATAGTAAGAAATTGG

425 1008 AATTGGCTGATGCAAAGAAGAA GACAAATGACTTACAGATCCCCC AAAAAGGTACATCAGAAACAGAAAC AAAAAGGTACATCAGAAACAGAAACGTGCTACTATCAGGGCAACTGAGCTT

426 1009 AGGTGTGAGCAGGGAGTGG GGAAATAGATATGGAATATTTCAAGAATCA TGTGCAGAGCTCTGAYGGCTGGTGA TGGCTGAGGCTGGGCAGAGCACAGGGTCACCAGCCRTCAGAGCTCTGCACA

427 1010 TTCCAGCTCTGGGACCAAG GTGCAGCCTCCTGGAGAAC GCCAGGCGTCCATCACTCCCTCTTC GCCAGGCGTCCATCACTCCCTCTTCCGGAGCTTCCATGGAATGACGGCCTG

428 1011 TTAGCAGTGGCAGGTTTAACATTC GCTAAGAGTTTTTTCTAGTGTACCAGCC AGTGTACCAGCCTCACAGCXTGATTCA CATTTATAATTGCTGAGTCATGAATGTGAATCATGCTGTGAGGCTGGTACA

429 1012 TGGAGTGCAATGGCGTGA CCTAGCTGCTTGGGAGGC TGAGGCATAAGAATCGTTTGAACCC TCGGCTCACTCCAACCTCTGCCTCCGGGGTTCAAACGATTCTTATGCCTCA

430 1013 GTGGTTGATCTTAGAGTATATTCCATGTG ACACTTAAGCAAATGGACCTAATAAACA AAAGAATGTGTATGTTGTGGTTGTT AAAGAATGTGTATGTTGTGGTTGTTCGGTAGAATGTTCAGTAGATGTTTAT

431 1014 AACATTTTCCAGCATAAACTCTTTTC CAGAACAGACAAGGTTGGGTG ATCACCTCATCTCACCTTCCTCTAA ATCACCTCATCTCACCTTCCTCTAACCACTCAGGATTCATGTCAGCCTAGT

432 1015 TCTGACTCCTCACCTCTTCTGC CAAAGACCCTGGGGTGGG CATTTTCCAAACATGCCTTCTTCCA CATTTTCCAAACATGCCTTCTTCCAGCTATTAAATACTTGAATGTACCGGG

433 1016 TCCTAAGTCCCTCAGCAGAAAAA TCCTTAGTTTACAATATTTACCATTAGTGTCTG GTAAGCAATTTGACTTSTATACAGA TTTGAAAACTGACTCTGGTGTGTACCTCTGTATASAAGTCAAATTGCTTAC

434 1017 TTTGAAACTGGATAATAGGCAGAGG GACTTCACTGCCCATGTTACTATCA TTGAAACTTCCTAGAAACTTGTTGA TTGAAACTTCCTAGAAACTTGTTGAGTGGTTGTGACCAAAATACTGATAGT

435 1018 ATGCACCTCCACTGAGCTG CAGCCACTACCTGCATGGG GAGXAGGACAGTGGCTTCTGTACTGCT GAAGGACAGTGGCTTCTGTACTGCTAACAGACCCCATGCAGGTAGTGGCTG

436 1019 CCTGTTTTTCTGTGGCTAGTGAG GTTTATCTGTCTAGTGACCTTGCAGC CACAGCTAGGGAAACAGGXTCTTCACA CTTATCTCTCCTCCTTTCCCAGGCACTGTGAAGATCCTGTTTCCCTAGCTG

437 1020 ACTGGACACACTACAGATTTGAACAA CTTTTAGCAGCTACATGCAAAGAAC CCCAAAGGTCTTTGGCAGTCCGTCT CCCAAAGGTCTTTGGCAGTCCGTCTGCTTTCTTTAGGGCCTGGAAGTTCTT

438 1021 AATGGTGGCTCATAATAGAAACTAAATTT AGCTCATTTTGGGAGTATGTATGTACA TATXTGTAACAGATTCCAACCACAGAG TGCCACTTCTTTTGCACATTTGAGTGCTCTGTGGTTGGAATCTGTTACACA

439 1022 AGACACACAGACCAATAGAACAGAATAG TTATTGAAGAGGCTGTCTTTTCTGC CTCAGAAAAAAATCCACACACCTAC CTCAGAAAAAAATCCACACACCTACAGTGAACTCTTTTGACAAAGGTGCCA

440 1023 GGTACCGGGGATCTCTGC TTGCTTTGGAAAGTATGGCC CACATGGAAAAACACTTTATACTAC CACATGGAAAAACACTTTATACTACGGATCAGAAGAATCAATATTGTTGAA

441 1024 GCAATTCTCTTTCTATGACAAGGTCA TGCCAGGAAATTAAAATCAAGTTAGA TTTCAGCCTCTGATTCCTTTATTAT TTTCAGCCTCTGATTCCTTTATTATCACAATTTTCTACTCCTGCTCTAACT

RθW# REF# upper PCR prime Modified Lower PCR primer Modified GBA primer Flanking sequence

442 1025 CACTGGCCTGTGAGCCAT ACCAAATGGCAACAACTACTCTG ATTGTCTTTTAAGAGAAATCAAATA TATAGATCAAAGAATTCACTAGAGCGTATTTGATTTCTCTTAAAAGACAAT

443 1026 ACTATGCATCCTGTGAACCCTG GTATCTTTATAGCAATGCCCCACC TAGATAAACACCTGAGATAGGGTAA CCCATTAAACCTCTATCATTTATAAGTTACCCTATCTCAGGTOTTTATCTA

444 1027 ATGTCTGAGAAGGTAGGATCTGGC CAGATACTCATACCTGTACTAGATTCCTCC AAAGACTCAGATCCTCATACCAGGG TCCATGTCTGAGGACGTAGAATCTGGCCCTGGTATGAGGATCTGAGTCTTT

445 1028 AGTAAAGGCTGAAAAAGAAGACTTTAATG GAAAGTTTTCATTTCTATCCCTGAT GAAAGTTTTCATTTCTATCCCTGATGCTGATCCATGCATCCATTAAAGTCT

446 1029 ATCCTTGCCTAAGCATCTGTGT GAAAAATGGACAGAAAKGAAGCTATT TTCGTCTTAAGACAGXACACACACAGA TGCATGTGTGAGTGTGTGTAGGTGCGTCTGTGTGTGTACTGTCTTAAGACG

447 1030 ATTTTTTTTTGTATTTTTAGTAGAGACGGG GGCTCACGCCTGTAATCC CAGCACTTTGGGAGGTCAAGGCGGG TCTCGATCTCCTGACCKCGTGATCCGCCCGCCTTGACCTCCCAAAGTGCTG

448 1032 CTGGTATTCCCTTCATGAGAATTTT AGGACAGTAGGAATGTTGGAATAGAC AATGCCTTGGTAAAGACAGTTTTCT AATGCCTTGGTAAAGACAGTTTTCTCCAAACTCCCTGAGAGGTAGGTCTAT

449 1033 TGGTTTCCTGTGTGGTAGTTCTTT GAAAAAGAAGAATTAGATTGGCACC TTCCGTCATCTGTTTATGTTATTCC TTCCGTCATCTGTTTATGTTATTCCGCACTCTTTYAGTTTTCAGAGCAGCA

450 1034 GTTCCCAAGGGTACTGGGTG AGGGTACATGGAGACCTGGG AGCTGAATTAGTATAXCTTACAAGTAT AAAGGGAGAGAGTCTTTATAATTTACATACTTGTAAGCTATACTAATTCAG

451 1035 TTTTGTGTGTGTGTGTGTGTGTG GAGACCTTTTATGTTTTGCTCTAACCT TGTGCCTGATCTCTTGTGTTTTGAG TGTGCCTGATCTCTTGTGTTTTGAGGTCCTAGTGAGGAAATGAGGTTAGAG

452 1036 TGTAATGGTCATGGACAGTGATG GACTTCACCACGAGGGCA TGGGAAGTAGTGAGTGATGGTGGGG TGGGAAGTAGTGAGTGATGGTGGGGAGCCTAGACCTCAGGTCATGGAGCCA

453 1037 TGCTCAGTAAGACCCCAAATTTT ATATAGTCGTTATAACAACAGCTTCAAAGTC CAGCTTCAAAGTCAAATAGACAATG TCCCRTGGCAGAACTTTGATTGAACCCATTGTCTATTTGACTTTGAAGCTG

454 1038 ACCCAATTCATACCAAATACTCTTCTG ATAACAACAGCTTCAAAGTCAAATAGACA AATGRGTTCAATCAAAGTTCTGCCA AAGACCCCAAATTTTGTCTAATCCCGTGGCAGAACTTTGATTGAACYCATT

455 1039 CAGGGCAGAAAATCATAACAATG AAAAATGCCTCCAACCTTTACAC TTCTTCCTAGGGCTGAATAACCAGA TTCTTCCTAGGGCTGAATAACCAGAGTGATTTAAGAGCTAGGGAATGTGTA

456 1040 CTCATCAAAAAGTGGGCTAAAGATAT ACGGCCATTCTTGCAGAA GAAGGTATACAAATGACAAAAAAGT GAAGGTATACAAATGACAAAAAAGTCATGGAAAAATACTGAATATCACTAA

457 1041 CAATGTAAGAGTTTCTACTCTCATTTTTCTG CTGAAGACCCCATCATAGTCTTTG TTXTCTCAGCATTXCAGGGGGTGAAAG TTTAGGGCTCAATGCTAGCCATCATGCTTTCACCCCCTGAAATGCTGAGAA

458 1042 CTCACCACTGCACTCCAGC CACTACCAGTTTCTTATGTATCCTTCTAAAG TCCTATACTTAAGAATACAAATATA TGTGTATTATTTATATGTAATTTCTGTATATTTGTATTCTTAAGTATAGGA

459 1043 CCTTTTGATGTTTGGGGTTTTC TGTTCAAGGACAAGGTATCCAAA AACAGGCTXGACTTAXTTCAATGAGAC TTTTCCCTGAACTGAACTGATATTCCGTCTCATTGAATTAAGTCAAGCCTG

460 1044 GAACTTCAACATGATAATAAAATTCACCA TGGGAATTCATTTGGCTTCC ATAAACXTAACTGAATAAGAGCAATAA AAACTTAACTGAATAAGAGCAATAACAATTTTTAAAAAGCTTAAAAATGTA

461 1046 CCAACTATAAACTTTACCTATGGAAAAATG TGGAGCACTTTAAAAATATTATCTCACTG AGTGTTGGGCCATTAXATGXTCTCTTA TGTTGGGCCATTAGATGATCTCTTAGTGCTCACCTATTTTGAGACTTCAGT

462 1047 AGGCAGAATCAAAGGCTTCT CGGCATGTAAATAACTTTTTAGGGA AACGGGCCATACTAACAAGTGCTGC GGATCTTTCCAGGCAGAATCAAAGGCTTCTCσCAGCACTTGTTAGT

463 1048 CATTAAGCCTTTCACTGAGAGTAGAGAT AGCTGGAAGGGTTTTGAAAGAT ACTTGCAGTTGCCTCTCTGACACAA ACTTGCAGTTGCCTCTCTGACACAAGTATATCTAAGAAATCAACATAATCT

464 1049 TTCAATAATGAGAGGGGGAGAAG CTCTCAGCTTTTTTTCTTGCTACTTG TGGXAGGXAGTGGGCTTCATATTCATT GAAGGAAGTGGGCTTCATATTCATTCGCACAGCTCCTCTCAACCTGGCCCC

465 1050 CGAAATGGCAATTTGTCTGAA CATATACACAGGTATTGTTTCCAAAGC AACATACTCTGCCCATTCCAGCCTT ACCAGCACTTAGGTTATCATATTGGAAAGGCTGGAATGGGCAGAGTATGTT

466 1051 TTGCTGTTTTTGAGCTCTATATTACATAAA GGCATTAGAAATTGGAGTAAAGGC TTGAGCATAAACAAAATCCAAACAA TTGAGCATAAACAAAATCCAAACAAGTTTTTTGCCAGTTGATAACATGGAT

467 1052 CTTCTTTGGCTATCATTTGGACAT TGGTATGAGTGTGTGTTKGTGTTTTA CCATTTCAAACATAATAGTCAAAGA CCATTTCAAACATAATAGTCAAAGAGAAGAAAATGCTCTATTCATTAAAAC

468 1053 GTATCAAAAGTGTCAATTCCCAAAGA ACACACACACACACACACACAATC CTACCTTTWTAATCTAXATTGACCCAC TATGGGATTAGCATCTAAGTGTCCTCGTGGGTCAATATAGATTAWAAAGGT

469 1054 AAGGTATCTTCTTGGCTCCAGTAAA CCAAGGTCACAAGCTAGTCTTAGC AGCACCAGGGCTAGGAGTTCAGAGA AGCACCAGGGCTAGGAGTTCAGAGACGTGGCATTTCATACTGGCTAAAGCT

470 1055 TTTGGAAGGCTCAGAAAAATACAG CAAAGACATTCAGCAAGTCTCTAGAAA GTCTTCAAAGCCCTCCAAGTCTCTA AAAATGTGGGAAAGTTTGGAACTTCCTAGAGACTTGGAGGGCTTTGAAGAC

471 1056 TATGACCAGTGCCTGTCTTAACTG TATTTGAAGAACAGAAAAGTCTAGTGAAGC TGAAGCCAGACAGCAGTAATCAGGA CCAGCTTCAGTGTGCACCAAACTCACTCCTGATTACTGCTGTCTGGCTTCA

472 1057 AGCTACTAAGAGTGCTGTGACTTTAAACT CCATGCAGTTAAAAAGAAACTCG TCGCCAGAGACTCACATCCTACCCA AAAATAAGTGGCAAACCAGTGGTTTGTGGGTAGGATGTGAGTCTCTGGCGA

473 1058 AGGCAAGGCTGAGTGCGC GAAAAAGAACCCTTAAGAAAAGATGC AAAATGAGCTTGTTCCCCTGAGGAG AAAATGAGCTTGTTCCCCTGAGGAGGTGTGACATCGCTGTCCACCCATCAC

474 1059 CTTTGGCTGTGTACTGATTTACACATAT CATTTTAACTGTTGGAATCACAAACA GCTCTTTAGCCTACTXCTTTCCAGCAG AAAAGTACCCAAGGCCGGGGGGGAAACTGCTGGAAAGCAGTAGGCTAAAGA

475 1060 GGAACTTGATATGTCTGAAAGCAAA TTCCGAATTTCTTTGGAAGATGT GATGTTACTGGTTGATTTCCATTTG AAGTGGAAAGTCTTTCAGAATAATGGCAAATGGAAATCAACCAGTAACATC

476 1062 TTGTTGAATGGTTGTAACCAAAATG CTTTGCTAAAGCATACCAAGAGTGA TACTCCAGTTCCCAACAAGTTCTGC AGGCTGAGGTGGTCTCAGCTGGAGACGCAGAACTTGTTGGGAACTGGAGTA

477 1064 CTAAGGCACTGGTTCATTACTACATAATG TCTCAACAAAAGGCTTTGATATGG GGAATAGTTGAGCTAACCAXTTCAGAG AATAGTTGAGCTAACCAATTCAGAGGCATTCTCTAGAATATAGTTAAATTT

478 1065 TGTGGCTCCCCTGAGTATTC GCTCAACTATTCCAATTTACTCATTATGTAG TAGTAATGAACCAGTGCCTTAGATT GAAAATTGTGGGCAGATATGGTGGTGAATCTAAGGCACTGGTTCATTACTA

479 1066 TATGTCAGGCCCTAGTATGTAGTATGAGT TATGAATCTGAAATCCCCATCATC AGGATATAAGCTCTCTGAGGGCAGG CAGTGGTGAACAGGACAGATATAGCCCCTGCCCTCAGAGAGCTTATATCCT

480 1067 RGGCATGGTGGCAGACAT CTCATTGTAGCCTCCGCCT CCTGGGTTCAAATGATTCTCCTGCC ATAATCCCAGCTACTCAAGAGGCTGAGGCAGGAGAATCATTTGAACCCAGG

481 1068 CACAGAGTTCCCCTCCCC GTTTCAACTTACAGGAAAATGAGGG GCTCTGTAATCTTGTCTGCGGAGGC GCTCTGTAATCTTGTCTGCGGAGGCGATTCCAGGCTTNGCTGGACGGCCCC

482 1069 TGCTCCTGCTTGAGCCAG GTAACCGCAGATCTTGGAGG CTTTCCCAATATAGACAGACGAGAG CTTTCCCAATATAGACAGACGAGAGGTGAGCAGGGTCTGCTTGATGAAATT

483 1070 CCAGGCTGGTCTCGAACTTAC ATAAAATAATTTTTAAAGGTCTGTTTAGGGC TCAGCCTCCTGAAGTGTTXGGATTCCA AGCCTCCTGAAGTGTTGGGATTCCAGGTGTGAGCCACCACACCCAGCCCTA

484 1071 TTTGGTTTCTCTCCGGTTGG GAATATTTTTCTTCTTGGGAGAGGC CXTGAAATATCAGATCCCXCATGAAAC TGAAATATCAGATCCCTCATGAAACGTGGCCATGGCTATATTTGCTACACT

485 1072 CAGGGGTCTGTGAGGTCAGA ACAAGAATATGTAAATACTCATTTCATTAGTCAC TCATTTCATTAGTXACAAAAGTGAT GAGACTACACGCCATGTAATCGCATCATCACTTTTGTGACTAATGAAATGA

486 1073 AAGACCCTGACTCTAAAAAGCAAAC CTATGTAAGGGTTAAGGTGGCTAGTCT GATTCGGTACAGGTTTTACXGCTCTGT CTGAAAAATGTGATTGGTACCAAGAAACAGAGCTGTAAAACCTGTACCGAA

487 1074 GTGGTTCATGCTATACCAAGTCATT TGAGAATTTGTAACACTTTTTGCTTAGTC AAGTTTTATTTTTGCCTTTGTTTCA GGTCAGCATATAGAACTTATTTTGCATGAAACAAAGGCAAAAATAAAACTT

488 1075 CTGCTGTTAGTCTAATGGGATTCC TCCAATTTTCCAAGGCTGAA TGAAATAAAAGAAAGTATGXTAAAGGC TAGGTAACCTGCCCTTTCTCTCTAGCCCCTTTAACATACTTTCTTTTATTT

489 1076 CCAACTTGTTTGCTTTCTCCC TGGTTCTCAAATAAGACAGACAAAAA TAAATTTXGCCTCTTTACAATCCCA TAAATTTGGCCTCTTTACAATCCCACATTTCTCAAAGGTTTTGTTTCTTTT

490 1077 AGTCTACTGTTTATTTGAGAAATGGCTT TAGTTATTTTCTTACCACTAAGGTTATAGGGATAT ACTAAGGTTATAGGGAXATGAAAGAAT ATGTGTATTTAGCTAAGAaTTTCTCCATTCTTTCATATCCCTATAACCTTA

Row# REF# Upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

491 1078 GTTTCTGGMACTTAATGATTTTTCAAA AGCATAAGGATGGCATAGGATTC GATTCAAGGATTTTTTTTTCTGTAA TTAGTTTCTTCTTGGATATTAACAGCTTACAGAAAAAAAAATCCTTGAATC 492 1079 TGGTGACTCACGTCTGCAAT CTGACTAATTTTTGTATTTTTAGTAGAGAATGG TCGAACTCCTGACCTCAAGTGATTC CAACACTTTGGGAGGCCTAGGTGGGCGAATCACTTGAGGTCAGGAGTTCGA 493 1080 AAAAACAACAGCAGCTCCACA GAAAGATAAGGAAACTCAGCTTTGG GAACATTGCAGATCACACAACCCAC TTCCCAATGCTCTTGAACAGGTCCTGGTGGGTTGTGTGATCTGCAATGTTC 494 1081 CTTGCTAGAAACTTGTTAAAYGGTTGT AGTTCCTTATTTCCACCTGAGACC GATCAAAATGCTGATAGTGXTATGGAC TCAAAATGCTGATAGTGATATGGACAATGAAGTTCAGGATGAGGAGGTCTC 495 1082 TGAATACAACACACACAATGGAC CTTAGACCGGCCTCGGAG TTCCACACCAGCGAAAGAGGGAGAC TTCCACACCAGCGAAAGAGGGAGACGCAGACAGGCCGCACCGGGCCTCCGA 496 1083 CTATCATGCAGTGAGATATATGGTTGTAA TCTAGACAAGTGTAATTATAGAAAGAAAGTGAAA CTTGGTATACTGCATCTCTXAAAATGG TGGTATACTGCATCTCTTAAAATGGGGGTTATTTGTATATGACTAAATAAC 497 1085 GAGAGGCACTAAACAGACACTTAAATT TCTTTAAGCCTCCAGCAGTGC AATAAAAACCTCAACTTTTTAGAAGATAAATAAAAAAAATCAATTAAATTT 498 1086 GGACATGGCATATCTTTTCATTTTT AGCACAGAAAATTAACTACTTAGGAAAAAA AGTTCTCTTCAATTTTCTTCATCAG 499 1087 ATTTGTATTTCAGATCCATCTAGACTCCT GGAGAGACTACGTGAGGGTGG TCTCTTCCTGGTGACCXTGTTGCTTCC TCTTCCTGGTGACCATGTTGCTTCCCCTCTCTAGGGTTTTCCCAGCCCCCA 500 1088 TGCAGTCCCAGCTACTTGG CTTGGAGTACAGTGACATGATTATGG GTGGAXGTGGGAGAATGATGTAAGCCT GGAGGTGGGAGAATGATGTAAGCCTAGGAGGTCGAGGCTGCAGTGAGCCAT 501 1089 GCATTCTCCGAGTTTGATTATGTT AAAATTCACAGTTTAGGCCAGTCA ATGAGAAAAAAAGTGTTXGACTCTACA GAGAAAAAAAGTGTTAGACTCTACAGGCATAAATATTGAAAAAACTGACTG 502 1090 TTCCCTGTACCCCACCCC CATAAAGCAGAATGTGGTGTGTG GCTCACTGGXAAAGATXCTTGGAACAT TGCTCAACATGTTCACACAATTCTCCATGTTCCAAGGATCTTTCCCAGTGA 503 1091 CAGCATTCGGTGAGAGCAA TAAAAGTTTGGAGCAAATAACCCA CAAAAGTAGCTGTCGGCTGAACACA CAAAAGTAGCTGTCGGCTGAACACAGGAGTTCATGTGTACTTTGTGACTGA 504 1092 CCTGTTTCTGGGCTCGACA TTTTTGCTCCTATTGTATATGTAGAAACTG TGTATCACCCCTAACAAGTCATTTT TGTATCACCCCTAACAAGTCATTTTGCCCATTGGAACTTCAGTTTCTACAT 505 1093 CGGTACCCGAGAGGATCTG TTCTTTGCTTTTTGTAGTGTAGGAAACTA CAAATCXCTGCTGTTTCTAAGTGAACC AATCACTGCTGTTTCTAAGTGAACCGTTATACTTAGTTTTATGTAGTTTCC 506 1094 TTYATAATAAGAAGGCCCTGGGT CTTGCTACCAGCTTGAGGCT CTGCTTCTCTGGGATTCTCTCATTC CTGCTTCTCTGGGATTCTCTCATTCGTACCTCTCTGTTCATGATGCTGCAG 507 1095 TAGTTCCAGCAGGGGCTAGAA CTCTTTAAGTGTCCTCCCTGGG TXTCCCTCTGAATTAGAAAXCTCTAGT ACCAGAAACCAGTTCATCAAGCTAAGACTAGAGCTTTCTAATTCAGAGGGA 508 1096 CAACCAGAGGATTCAACAGAGTT CAGCGTGGTGCTTCATGA CCTGGCCACTCTCGCAGAATAGTCA AAGGAGCCTGACGGTTTCACAGAGTCTGACTATTCTGCGAGAGTGGCCAGG 509 1097 CTTTCTGATTGTTACACTTTTTTTTTCC CAGGGGAATTGCTTGGACC CXAGGCTGGGGTGXAGTGGTGCGATCT AGGCTGGGGTGCAGTGGTGCGATCTCGGCTCACTGCAACCTCTACCTCCCG 510 1098 TACCGGGGATCTTATCAATAGTGA CATGAGACCTGCCAGGAAAA CTTTGGTCTGACCTGAGAGXTCATACC GAGTCCAGCTCCAGGTTGAAGTCCTGGGTATGATCTCTCAGGTCAGACCAA 511 1099 TCAGGTCAGACCAAAGGCA CTTGAATCCTTGACTGCACCTC ACACTACGATGTGGGGAGGACAACT TTTTCCTGGCAGGTCTCATGGGAGAGAGTTGTCCTCCCCACATCGTAGTGT 512 100 CTTGTGAGTTCAATTTTTTTCTCTCTC TTTTATCCTGGGTTTCTAAATTATTGC TAAATTATTGCXGTAAACTGATTACAT CTTCAAAGAAGAGCCCATATGGGAAGATGTAATCAGTTTACTGCAATAATT 513 101 ATTAGAAACATCCTTCCCAGGC GTTTTGTTCACTTGTCTCAGAACATG GAAAACCAAGGGAGAGAACAAACTC GGACTATCATCATCCTGCTCCCTCTCGAGTTTGTTCTCTCCCTTGGTTTTC 514 102 GGCATCCCAGAAGACATCTC TTTTATTCCTAGAAAATCCTCCTTTCC TCCTCCTTTCCTTCTAXCACTCTGATC AGAAGATAGCGTTAACATATCTAAAAGATCAGAGTGATAGAAGGAAAGGAG 515 103 CGGGGATCTTTTCCTTGTATTA GTTGTTAGACTCGTGGGACGAC CACTGTTTAAGGGTAGTTGTGGTGG TTTGATAGCCTSTGACACTAAGATGGCCACCACAACTACCCTTAAACAGTG 516 104 TGCCTCAGCCACCCAAAG TTCAGTTCACTTGCCCACTTTT TGCTGXGATTATAGXCACGAGCCACCC CTGGGATTATAGGCACGAGCCACCCGTGCACAACCAACAATTCATTTAAAA 517 105 AACGTGGAAAGGACCACAGC CTTCCACACAGGTGTACCCAC AGGACACCTGAGCTCTCCAACAGAC AGGACACCTGAGCTCTCCAACAGACGTTTAACGCCAAGGGATTCACAGGTG 518 106 CTAGCATTTCTCTGAAGGATAAAACAAA AGGGYCAATGGAGATTGCA TTTTAATTCATTGAAGTTATTTTGG CAAACAAACAAATTAACAAGAAAAAACCAAAATAACTTCAATGAATTAAAA 519 107 AATCTACAGCATCAGAAAGACTTGTCTC CTACTTCTGCCTCCYAAAGTGCT AGTXCTGGGATTACAGGGATGAGGCAC AATAATCAGAGCTCTGGCCAGGCGCGGTGCCTCATCCCTGTAATCCCAGCA 520 108 CTAGAGTTGTTTCAAAGCTAAAAATGGT TTTCTCTCAGTTTGTAGGCTCAAAA TCCATACGGGGCTAAGTGAAATAAG TCCATACGGGGCTAAGTGAAATAAGGTTCCACCTAACAATCTCTAGTTTTG 521 109 GGTTGTGTGTTTCTAAGGATTTGG ACCAGAATAATTCTAACCAGAAAGGAA AGAAAGGAAAAAGAGGCATTAAAAC TAATACATTTTTAAAAAATAGAATCCGTTTTAATGCCTCTTTTTCCTTTCT 522 110 CTGACACATAAAGAGTCCACACATTT TTGTTGTCCTGGTTTTCAATAATTT ATAATTTGGTTCTATTTTCTAACAT AGCATGCAACAAAAAAATTACCAGGCATGTTAGAAAATAGAACCAAATTAT 523 111 CCTTTCACAACACGAGGGG AAGAGATAAATAGGAATGTGTATGGGAA GTATGGGAAATTGTGAAGTAAAAGG AAACCATATCACATTTTTAAAAATTGCCTTTTACTTCACAATTTCCCATAC 524 112 ACACGTTGGAATTATGGGAGC CATGTGGTCTTGAAGGGGTCT GTAAGATTXGGATGGGGACACAGCCAA AAGATTTGGATGGGGACACAGCCAAACCATATCACCTCCATTGAGCTTACT 525 113 ATTCCCCAGCCATGCTAAAC TACATTCCAGCCTGGGCA AAAAAATAAAAGTAAAGACATACCC CTTTCCTTTATAAATTACCCAGTCTCGGGTATGTCTTTACTTTTATTTTTT 526 114 GTGGGGCTCAATACTCATGCT TGTGACTAAGTAAAAAACATTGAGATTATCC AGATTATCXTGATTTATCXTTATAGGT TTCAAGAGCTTTTATGATCACATTACACCTATAAGGATAAATCAGGATAAT 527 115 GAGCTATCTATGACAAACCCACAGA TGGTGAGAGTGGGCATCC CATCATAACGAATAAAATCTGGAAG CATCATAACGAATAAAATCTGGAAGGATTATACTTGATAACTATAATAAGA 528 116 GAAGAGGCATTCTATTTCAGCAAG TGCAGCATAAAAGCAAGAGGG AGTGGCAGGAGAAAAACTCAGGTTA GGTTTTCAGAAAAAAAAAAAAATAAGTAACCTGAGTTTTTCTCCTGCCACT 529 117 CATTTGTTAAGTCTCTTTACAAGGCC TGGGCAAGGACACAGATG GCAXACCATACCATGATGTTTACTTTT TGTTCCTTTCAKAGTCAGCAAATAAGAAAAGTAAACATCATGGTATGGTTT 530 118 GTGGACATTCAGTACATTTGTTAAGTCTC ATGCAAACCATACCATGATGTTTAC TGTTTACTTTTMTTATTTGCTGACT TTACAAGGCCCATGTGTTCCTTTCAGAGTCAGCAAATAAKAAAAGTAAACA 531 119 GTCTCTCTTTCTGAAAATCATAGAAGGAT TGCAGAACATTCTTTTTTTGGC AAAAGTTCCAAGTCAAACACATTCA AAAAGTTCCAAGTCAAACACATTCAGCTTTCATCCTTTAAAAATGTCATTA 532 121 CATCTTTTTCTAGTTGTGGCTTATCTTT TGGGGAAAGTGATGAGGATTT TCACACAATTXAAGCTGATATTTAAGA ACACAATTTAAGCTGATATTTAAGAAACAAATTCTTAATTTAAATAAGATT 533 123 AGGTGAATGGGAAGCAGACATAT GTACCGGGGATCTGGTTOTT GGTGGATCAGGAGAGAGAGAGTGGA GGTGGATCAGGAGAGAGAGAGTGGAGGGGGAAGTGCTACACACCTTTTAAA 534 125 GCAACAAACTTGCCAGCTC GCTGCTATAACAAAATATCTGAGACTGG AAAXAACAGAAATGTACTTCTCACAGT TTGATCTTGGACTTTCAGCCTCCAGAACTGTGAGAAGTACATTTCTGTTCT 535 126 AATTCAAGATGAGAATTGGGTGG GAATTRAAAATGAGACAGCCAGC GATGAAGCTTAGGTGACAGATTTTG GATGAAGCTTAGGTGACAGATTTTGAACATGAGACCAATCCACCCACAGAG 536 127 CACAGGCAAGTAATTCATTTTGTG ACAGTTTTCAAGGTTATAAATGACTGTCTT ACTGTCTTCCAAAAAAAXCTGATGACA 537 128 TCCTAGATGACCCCTGGCTT CGGGGATCTGCTCGGGTT GGCTGGTGAGCCTGGGGACTTGGTG GGCTGGTGAGCCTGGGGACTTGGTGGGAACTTCTCTGAGAAAGGGAAGCAG 538 129 CCCGTATGGAATTTCAGACTACTTT ACAAGTGATGGTTAACTACTAAAAACTGAATAA CTAAAAACTGAATAAGCATCTCCGC TTTGCCTCTGGGTCTTTCACTGAAACGCGGAGATGCTTATTCAGTTTTTAG 539 130 TGTGTGTGTATGTGTGTGTGTTGG CAAGAACACCTGCCCCGT AGAGTTATTGAGAXAGCCAGACTTTTT AGTTATTGAGAAAGCCAGACTTTTTAAAAAAAAATGCAAATTTCCAGGCCT

RθW# REF# Upper PCR primer Modified Lower PCR primer Modified GBA prime Flanking sequence

540 1131 ATTAAGATGCAGTGGGAAGAGCT TTATTGTCTATCCTTCTCTCCATAAAATAAAG TTTAGAGTXCCXGCTGTGTXCCAGGCA TAGAGTGCCTGCTGTGTGCCAGGCAGTTTCTAGATGCAGTAGGTATACGTC

541 1132 AACATTGGTGCACTGGTACCC ATGATCTTTCTAATGCTTTCCATGAA AAAGACTTAGACAGCCACACAATAA AAAGACTTAGACAGCCACACAATAACAGTGGGAAACTTTAACACTTCATGG

542 1133 GTAGGAGTCCTTTCATTTTATTCTTCTTTT TGCCATTTCTCCCCCAAA CAATTTCTACAAAAAAACCATCTTG CAATTTCTACAAAAAAACCATCTTGGATTTTAATTTTGATTTCAATTAAAT

543 1134 GTTCATACTTAGCATAGTAGMTGATAGGTAAT TGGAGGTAGGTCTCTATGACATGC TTTAATAAATTGGTCTTCTACCCTT TTTAATAAATTGGTCTTCTACCCTTGTTTCATTCCTCTGGCTTCTATGATG

544 1135 CCACTTCACACTTTTTGGTATTTTTT AGTTGCTAGGTTTGGGATACTTTTAAA AAATACAGCAATAGATACCAAATAT TCACTCCACTGATGGTACCAAAACCGATATTTGGTATCTATTGCTGTATTT

545 1136 ATTTTTGCACTGGTTATTTCTCTCTG CAYTGGGTAAATACAACCATTTCAA AGAAATTGGXCATAACAAAGAGGCTAC CTGGATTTCGGATTTGCATGGGCCCCGTAGCCTCTTTGTTATGGCCAATTT

546 1137 GATTGCTTGAGGCCAGGAG ATAATAAACCACCCAGTATAACCACATATTTA CATATTTAGTCCTXTTCATTAAAACAA TTAGAAAAACCTATCTCATCAATTCGTTGTTTTAATGAAAAGGACTAAATA

547 1138 ATCTCCACACCACTCTCCTGATC GAAAGATGTGACAGAAAATACTGATTAGG GGAXTGCATGTACACAGTTTGATGGAC ACTGCATGTACACAGTTTGATGGACGTCTGTGTGTGGCTGCCTCATACAGA

548 1139 GATTTGAGTGATGAGATATAAGTTATTTTAAGATG TCATTAGTGCTTAAGTTTTGAAATTGTG AAGCATATTGGATTGGXTACTTTAGCA TTTCTAGGTAAGAAGAAGAAAAATTGTGCTAAAGTAGCCAATCCAATATGC

549 1140 CGGGGATCTGAAAAGCCA TAAATGTGTGGTGAACCACGG TTTTCAGTACAXAGACAGTCCTTTAAC GCTTGACTATCTGCCAAGTTTTTAACGTTAAAGGACTGTCTTTGTACTGAA

550 1141 TAATCGTTAGCAYCTTCCAGAATTC CTCTACTACAGGGAGGCTCACAAG ACAAGATAAATTOXCTCCCTTTGGCTG TGTTTGTGTAATTGAAGAAAACAACACAGCCAAAGGGAGGCAATTTATCTT

551 1142 GACTCTGAGCTTGAAGAACCTCC CTTCAATTACACAAACAGCAGCC TGATAATXCAAGTXCTXATCGTTAGCA ATAATGCAAGTGCTAATCGTTAGCACCTTCCAGAATTCTTGTAGTTCTCAT

552 1143 CCGTATGGAATTTCCTAAAAGTATAGAAA TGCCGACATAATGCTGGG CCTCATGATCTGCTAACTCCACTTC CCTCATGATCTGCTAACTCCACTTCCGGAAATAAAATCAGTATTTTCCAGA

553 1144 CATTTTTTCTGTCTAATCTGCCCTTA TAAAAGTGACCCAAATGAGAGTTCA TACCCAGGTTATTTTTTATTTTAGT TACCCAGGTTATTTTTTATTTTAGTGATTGTAGTTGTCATTGGTTGAACTC

554 1145 GCCAACATCTATTGGGTTTTTTTT CCAGGGAAATGCAAATTAAAATG CAGTGAGATACCACCACCTTATTTC ACTTTTTAATAATGGCCATACTTGCAGAAATAAGGTGGTGGTATCTCACTG

555 1146 AAGCAGGGAAACTCCTTAGCTTT CATTCTAAATAGCTACTTTCCAGGGG AGAGGTCCATCTGACTGTTGATTTG AGAGGTCCATCTGACTGTTGATTTGCGATATTTAGTCTTCCTATTACAGTT

556 1148 ATCTAAGGAGCTCTGTAAATGACTATCC ACGTTGAAGTCAGTACGCAGG AGGCTGGCATAGGCXTGATACCTGG CAAGGTCCTATGTGTTAGTCCTTGCCCCAGGTATCATGCCTATGCCAGCCT

557 1149 AAAAATGTACTGAGCTTCTTGTCACATAT GATGAGTCCAAGGTGGGCA CAGXGTCAGATCXTACAGGTCCCCTTA ATAGAAATCAGGTCCCTTTGCAGGCCTAAGGGGACCTGTAGGATCTGACCC

558 1150 CAGAATATACATTCTTCTCATCTOCACA ATAGTTTTGACTGTTCCTCTTTGTGTTC TCAAGTACTTTCTCAGGTCACAGGA TCAAGTACTTTCTCAGGTCACAαGAGAATAAAACTAGAAATGAACACAAAG

559 1151 AAATGGGCACAAGGATGG CTTTCTCATGGATCTTGGACTTCT AGGGCGTTXCACACAGGGCATTGTCTT GGCGTTGCACACAGGGCATTGTCTTACCAGAGGCAAACACTCTGGGCTGTG

560 1152 GAATGAGTCAGGATATAGCAAGTCTATAATACA TGGAATTTGTGAAAATGCTAACTTG TAGCAATTCAAACAGAGGGAAAAAA TAGCAATTCAAACAGAGGGAAAAAAACTATAAATTGCAGAAAATCTACRTT

561 1153 TACCGGGGATCTGCTTAGC GAGCTCCCACAATGA CATGG CTACACTGCACTGCTCTXTAAGAAACT AAGCATTAAGTTTGATTGTTGTGCAGAGTTTCTTATAGAGCAGTGCAGTGT

562 1154 ATCCAGCTATAATCCTCATTGCAC GACAACCAAGTATCTCAAAACCCA CTGTGTTGXCCTTCAGCCTGTTAGACT GTGTTGGCCTTCAGCCTGTTAGACTATTTTACGTAGGACTGAACCAACCCC

563 1155 ATAAGAAGGTGAAGCTGTAGGACCA CAAATACAGGGAGATTTTCTAATGATTTT CAAACAAGGGAGAGAAAGGGAGTCC CAAACAAGGGAGAGAAAGGGAGTCCGAGGAATTGGGGACCTGGCCTAACAA

564 1156 TTATACTCTAATTCCTAAGCATCAGTAACACA GGGGATCTGTTGAAGTTCCTCT CTATGAAAGTTAACAGAACGTAGAA CTATGAAAGTTAACAGAAαGTAGAACTCAGCTGCCCCTGCTCACAAGCATC

565 1157 ACTGTTGTACCCAGGAAGCATG TAATTATTGCCGTCATCACCCT AACTATGTTXAAGGGCTXAAGATTCAC TGCTGACAGAAAAGAAGTTTCAATACGTGAATCTTCAGCCCTTCAACATAG

566 1158 TGGGTTATAGTCAAATGGTAAGGC GTATTTAGCAATTTCAAAAAGTAGACCCT CTCCATCACTGGCTGTTGXAGTCTTCT AAGCAAAATACCCCAATTCTGAAAGGAGAAGACTTCAACAGCCAGTGATGG

567 1159 TACACTGTGATCTGAGAAGATATTTGA CGCTAATAGACATCTACAGAACATTCC ATATTATTXCATTGTTTTGXATTTATT ATTATTTCATTGTTTTGAATTTATTGAGACTTGCTTTATGGCCAAGTGTTT

568 1160 CACAAAGCATTAGTGACTTTAGTGACG AGTCGTTAATGAGTGATTTTAAATAGCTTG TAAATAGCTTGCTGTGTGCTTCCTA TGAGGATTATTGGCCAGACTTCCTACTAGGAAGCACACAGCAAGCTATTTA

569 1161 CCTACAAAAGCAATGCCCTG CCAGCCCCGCTAAACTGT TCTCACACTCCTAATAAAGACATAC TCTCACACTCCTAATAAAGACATACCCAAGATTGGGTAATTTATAAAGAAA

570 1162 CAAGATTAAAAGAGAAATCGTTGARAATAC CTGCTGCCCTGTGAAGAAGT CCATTATGATTGTAAGTTTCCTGAG TCACAGTTCCACATGGCTGGGGAGGCCTCAGGAAACTTACAATCATAATGG

571 1163 GGATCTTGGGACGCTGTG CCAGTAATGGAATTGTTGGCTT GXAAGCTGTXTGGAARTTTCACAAAGA AAGCTGTTTGGAARTTTCACAAAGAGTTTAAAATAGAATTACCTTTCAAGC

572 1164 CTGATTTCACTTATGCACATTTAAAGAA TCCACAGTGCCATCCACC TTTCCCCACCAAAAGACAACTAATA AAGTATTAAAGTACACCAAATTTTAGTATTAGTTGTCTTTTGGTGGGGAAA

573 1165 CGGGGATCTGGTTAAATGG CTCTGCTCCGGTCTCTGG TGTCTCTGTTTGCTCCXTGAGATGCGA TCTCTGTTTGCTCCATGAGATGCGAGCCCTGAGAGGCCAAAGCTATTTTTT

574 1166 TCACTGTCACGAGAATAGTATGGG CTCGAAYTGTAATCCGCATGT GAAACTGCCCCCATGAGCCAATCAC GAAACTGCCCCCATGAGCCAATCACCTCCCACCAGGTCCCTCCCTYGGCAC

575 1167 CAATCACYTCCCACCAGGT CTGATATGGTTTGGCTCTGTGTT TCCCTCCCTYGGXACXTGCGGATTACA CCTCCCTYGGCACATGCGGATTACAGTTCGAGGTGAGATTTGGGTGGGAAC

576 1168 TAATATCAAGAGGAGCTGCTGTCTTC CGTCCCAACAAATCACCACT ATTCACTCTGAAAACTTACCTTTGT ATTCACTCTGAAAACTTACCTTTGTGGACTCTTTGCCAAAGGTCTGTTCAG

577 1169 CTGGGAACGGTGAGGTGA AGAGGCCATAGTTTTCAAGTAAGTCTT CTTCATTTTCTTTCATTTTCTGTGA CTTATGTTTTGAATTCCCAAGGGCTATCACAGAAAATGAAAGAAAATGAAG

578 1170 GGTATCCAGCAGATATCATCATTATTA AGAAATCCTTTTTTCCTCAGCC ATTGTAGATGTTGTTATTXTAGCCCAG TGTAGATGTTGTTATTATAGCCCAGACAAGGGGATCTAGTGGGAAAGTGGC

579 1172 ATAGCAAGTCACCTTTACTCCAGTTTC GAAGATGTGGGAAGGTTCCAA AGACCACCTCTGCCTTGACTTCATT AGACCACCTCTGCCTTGACTTCATTGTCCATGTCACTATCAGCATTTTGGT

580 1173 ACCACTACGCCTGACTAATTTTTG CACTTGGGAGGCCAAGGC TATTTTTAGTAGAGATGGGXTTTCACC TTTTTAGTAGAGATGGGGTTTCACCGTGTTGGCCAGGCTGGTCT GATCCT

581 1174 ACACAGACCCCCCAGAGG CTTGTCCAGACTCCCGGC ACCTAAGGAGAACATXGGAGAGCCCAA CTAAGGAGAACATGGGAGAGCCCAAAAGTCTCCTTGATTTGAGGAGACAGA

582 1175 TATGGTGACAGAGAGGTCAGCA CAAGGCAATAATTGAGAATGCAA AACCTTTCAATTAAATAGGAGAAGC GGTGGGGGCAAGAACTCTTGAGATGCGCTTCTCCTATTTAATTGAAAGGTT

583 1176 GCAATGACATTCCTCACAGAAATAG ATTCAGGTAGTGTAATGTTTCCAGTTTT TTCCAGTTTTGTTCTTTTTGCTCAG AAAGACCCCAAATAGCCAAAGCAACACTGAGCAAAAAGAACAAAACTGGAA

584 1177 CGGTACCANNGGGGATACTTTT GCTGCCTAACGTGATGATAAGAGT CAAAGAAGCTGAAAGAAAAAGGTAG AGACCCCNCTATGGACATCCCATTTCCTACCTTTTTCTTTCAGCTTCTTTG

585 1178 AGGCCATTCCCCCACCTG GTAGAAGGCTCACAGCTTTTGTG TGTACAATGCCXGTCCCCCACCACAGC TACAATGCCTGTCCCCCACCACAGCGGTAGTGCTGCAGTAGATTTTTGCTG

586 1179 CCAGGCAGAGACTTATTCTCATCTAG TGGCTACACTATACAACAGATGTTCAA CTAGATAACATCTTCTTCCAATAGA CTAGATAACATCTTCTTCCAATAGAGGGCTGTGTTGTCTACATTGAACATC

587 1180 AAACAAACATCCAGAGCCCTG GTCTGCCTCCTCTGCTGC TGGGGTCTGCCTCCTCTGCTGCCTC GCTGCTCCGGTCCCAAGGAGAGGCTAGAGGCAGCAGAGGAGGCAGAC

588 1181 AACTGTGAATAAATTGAGCCTCTTTATC TGGTACCTGTAAACAAGAACCATACC AGACTCTXGTRAATACAATACTCTTGT ACTCTTGTRAATACAATACTCTTGTGAACACAACCAATATATTTCTGGTAT

Row# REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

589 1182 TTGAATTGTAGCTCCCATAATTTACAC CTACCAAAAGAATAGCATGGGGA GGAAAACTGCCCCACAXTTCAATTC GGTTGTGGGAGGGACCCGGTGGGAGGGAATTGAATTGTGGGGCAGTTTTCC

590 1183 GCGTTAATACTGATTCATTTATTAGCTCTAG AGAGATGATAGTATCTTGCAGGTAGAAAAC TAGAAAACTCTAAACCCTAAACCCT AGTTTTGTGYGTGAAGGGGGATCTGCAGGGTTTAGGGTTTAGAGTTTTCTA

591 1184 TGAGCCAAACTGCCTGAATG TCGAAAAGGAGGGGGCCT TATTGCCTGTCAGTTTTGTTTCAAC TATTGCCTGTCAGTTTTGTTTCAACGGCTCTTCATGTTTCAGTTCTTTTGC

592 1185 TTTTTTCTTTCTCTTTTTGTAGGTATGG GATCATTTCAGCCCAGGAGTC TTTGCCGCXCAGGTGGGGTXCAGTAGC TGCCGCCCAGGTGGGGTGCAGTAGCGTGATGATGGCTCACTGCAGCCTGGG

593 1186 AACAGTCTTTCCATATGTTTACCTCC CATTTTGTCTTCTCACTGCACATC TCCXTCXTTCTCAGGAAGCCAGTCATT ACCTGTCATGTAGTTGAAGTACCAGAAATGACTGGCTTCCTGAGAAGGAAG

594 1187 CTGTTTGTGTGTCTGCTTTTATGC AGCCAATGCAATTCTGAGCA GAAXGAATATGTAGGCATCATACTACC GATGTTTAATAGCTTGTAGTATATCGGGTAGTATGATGCCTACATATTCAT

595 1188 GCTCGGGGATAGTATGTGCA ATTCAAATCTCTTGCCCATTTTTC TTCTATTGAGCTGCCTTTTTCTTAC AGTCTACAAAAGATATTTATAGATAGGTAAGAAAAAGGCAGCTCAATAGAA

596 1189 ATCTCAGCTCACTGCAACCTC CATAATGAAACCCTGTCTCTACTAAAACTAC GCCTCCCACGTTCAAGTGATTCTCC GCCTCCCACGTTCAAGTGATTCTCCCGCCTCAGCCTCCCAAGTAGCTGGGA

597 1190 GAGCTGAAGAACCATCTCTGCTAG ACCCAGGGGATCTAATCTTTTTT AATATTTAXAGATATTTGATAACTTTC GTATGGCATGTCAGCATACGAAAGTCGAAAGTTATCAAATATCTTTAAATA

598 1191 AACATAGGGAGACCCCCCC CAGAGATCCTCCTGYCTCAGC TAGGACTGCAGSCATXTGCCATCACAC CCAAAAATAAAAAAAAAAATAGCTAGGTGTGATGGCACATGSCTGCAGTCC

599 1192 TGAGGCACTTTGGTATTTCTCAG GAATTACTCAGTCTCGGGTATTTCTTC CAGTGTGAGAATAGACTAATACAAG CTCTTGTCCTGAGCACATAGTAGGTGCTTGTATTAGTCTATTCTCACACTG

600 1193 CCTGGCACCATTATACAAGCTACT CTGTTGCTGTTGAGGAGGGTT TGCAAGCTXTGTGTGCAGAACCCAAGG CAAGCTCTGTGTGCAGAACCCAAGGCGCTCTTCTGGAGTGAACTCCACAAC

601 1194 CACCAATTACCACAATTCTTGCA ACGATGCTATTGTTATTTCCCAAGTA TTATTACCCTCGTTGTCTTCTCTCT TTATTACCCTCGTTGTCTTCTCTCTGTATATCAATTCTTCAATTCTTCAAG

602 1196 CCAGACAAAATTATTACCTGTTTCCA TCACCAGGATTTTCAACTGAGAA CTGTGTCTCCTGAGTGTGTCACCTC CTGTGTCTCCTGAGTGTGTCACCTCGTTAGGTTCAATTATTCTTAAATTTC

603 1197 CACTTCACCTATAAAGACACATATAGACTTAAAG ACCTAATATAAGTATAACAACTTCTGCTCACTCT CAACTTCTGCTCACTCTXGTTTCCATT GAAGGGATGTGAAAAGACTCCACACAAATGGAAACCAGAGTGAGCAGAAGT

604 1198 TGTGGAGTGGTAGCAATGCTC GTGAGGGTGAAAAGGTAGCACA AAGTXACCATTTGTAAGAGAGTAAGAA GTTACCATTTGTAAGAGAGTAAGAACGGATTATTTGGTCCTGTCTTCTCTG

605 1199 GGCATAGAAACAGACACATCGAC TTCTTGGCACCTTTGTCAAAA CCAGAGAGAAAGCCCAGAAATAAAT CCAGAGAGAAAGCCCAGAAATAAATATAGTCATTTGTGGGCAATTGATTTT

606 1200 AATATCCTGTGTTCATGGATTGGA ACTGTTAGGGCCTTTTGTAGTTCC TTGAATTTTGGTTAGGGATTGTATT TACTACTTCAAGTGATCGACAGGTTGAATACAATCCCTAACCAAAATTCAA

607 1201 TGCAAACATTCCTTCTATGGCT GAACATTCCCTTAAGCCAAAGC AAGCCTAATCCAGAGCAAGACCCTA TCACCTCTCTCAGCCTTCAAGAGAGGTAGGGTCTTGCTCTGGATTAGGCTT

608 1202 CCTTACACTTTTATGTTATGAAAACAACTTG TGAAGGCTGAGAGAGGTGAGTAAG CCTTAAATCTCATGAACCTACCTCT CCTTAAATCTCATGAACCTACCTCTGCTAGTTGCAAACATTCCTTCTATGG

609 1203 ATTAGGTCACGTCACATGGCA GAATCTTTCTTGAGCTCCTAATCCC CCGACCTCTACTCCCAGAXXTAAAGGA AGCTTAGCCAATGGGCATGCCATAAGTCCTTTAAATCTGGGAGTAGAGGTC

610 1204 CCATTGAGTAAATAATGCGATTGTC AGTCTCAGATATGTCTTTATAACAGTGTGG TAACAGTGTGGAGAATGGACTAATA TATGTCATAGTGGCCCTTGAAGATTGTATTAGTCCATTCTCCACACTGTTA

611 1205 GCAACCTACGCAAGAGATAGGA TCCACACCTCTTCATGCTAGAAA GGTAAAGAGAGAAACAAAACTATTT GGTAAAGAGAGAAACAAAACTATTTATAACTCTTATCAATAACATTTTTCT

612 1206 TCTGTCTATGGAGCAGCCATTT CTGAAGAGAGGGAAGGCATG TCTTTCACTCTTGGCTTGCTCTTGA TCTTTCACTCTTGGCTTGCTCTTGAGTTGTTTCCTGAGTGAAGCCAAGAAC

613 1207 AAGCTGAGCCCCATGCCT CTCCTTCCTTCTACAATCCCTTAA TGCTGAGTGGGGAATGAGGTAGGAG TGAGATGGTGCAGCTATCTGCTTCACCTCCTACCTCATTCCCCACTCAGCA

614 1208 GTATACATAATAACATTCTCTATCACCCAA TGTGTGTCGGGGTTGTTG GGTTGGGGTTOTTTGTTTTGTCAGG GGTAAYGGAGGGAGTCACCTTTCCAACCTGACAAAACAAACAACCCCAACC

615 1209 GGCTAATTGTTCAGAGATTTATTTAACTAGC GGGTTGGGGTTGTTTGTTTT ATAACATTCTCTATCAXCCAAAGGTAA AACATTCTCTATCACCCAAAGGTAACGGAGGGAGTCACCTTTCCAMCCTGA

616 1210 TCCCTCCACTCCTCCAACC CTAACCCCTCTGTTCGATCTTTATAATAC TCATGAGAAAGATAAGCACTCCCAC TCATGAGAAAGATAAGCACTCCCACCACCAAGACCTGGAGGTGGGAAATGA

617 1211 TCCACTGTTCATGGGCACC TGCATGTTCATCGTGGCA GGCTGACTCCATGTCTTTGCTATTG GGCTGACTCCATGTCTTTGCTATTGGTCCATGTCTTTGCTATTGTGAATAG

618 1212 ATTGGTAGGAGGAGGACTGGAG GATCTGAGATGGATCTTTCTTTGC CCTTGAATATCCGGAAATCTATGGC CCTTGAATATCCGGAAATCTATGGCCGTGGGATGCAGCTGGAGAGAACTGT

619 1213 GGTMAGGGTAGATGAGAAGKAGTCTC AGNCGCTTTGCTTTTTGTT AGAAGAACAGAAATTCATAAAATAA AGAAGAACAGAAATTCATAAAATAAGCTATAACAATCAGGTTAAACAACAA

620 1214 GGAATAAATATTATCTCATTTGATAGAGTGATTG GAGTCCTCCCCAGCCATG WTTTTCTTTTGTAAGTTGTCCAGTC TTAACACTGCTGATAAAGACATACCCGACTGGACAACTTACAAAAGAAAAW

621 1215 ATGGGTAGCAGAATCAGACTTCTTT GTCCTGTTATCCTTTTCAGAGTCTTAGAT TAGATAAGGXGCTTTGCTCAGCCTGTG GCACACTTTCCCGTTGCCTCAGCGCGCACAGGCTGAGCAAAGCTCCTTATC

622 1216 CAGGACTGGGTTTTATGTCTCC ACCGGGGATCTTTTTAGTCTATG GATXTAATTAAAGATAGAATGGTTGTA TTAACTATTAGATAATTAATATTCTGTACAACCATTCTATCTTTAATTAGA

623 1217 ACACCATCAGCATCAGCAAGA CCCAAATATCTGTTGGATGAATAAAG AATTTAGAGGAGAAAGACATAGGTG CCTCCTTGAGATCCTTTTGTTCCTCCCACCTATGTCTTTCTCCTCTAAATT

624 1218 ACTGTTGGGAAGGCATAATTGG CTCCCACAATATGTGATAATTCAGG GTTTTGAXATGTGAGGACXTGAGATTT TTTGAAATGTGAGGACATGAGATTTGGGAGGGGCCAGGGGCTGAATGATAT

625 1219 AGAACAGACTAATACAGTCAACTGGTACC CCTCTGCCTGTTTCCCAGTT AGTTCCXAAGTCACTTCCXTGTTTTTG GAGTGGGGCRTTGCTGAAAAGATACGCAAAAACATGGAAGTGACTTTGGAA

626 1220 GAGTTATTCAACATTACCATGAAATGC TGGTTCCTACAAATGAAGACCAAT GCTTATCTGTTCGTGGTAGAAGTGT TTTTTTAACAATCCTACCAATTTACCACACTTCTACCACGAACAGATAAGC

627 1221 GTTAAAGTGACATGTTCAGGGTCAC GGTCCAGCGGAAGAAGGTT GAAGGGAXAAXCAGAAGTAAGATTCTA TACCTAATTTGTTGCAAAGGTAGAACTAGAATCTTACTTCTGATTCTCCCT

628 1222 CAGGGGATGACATGGCAA CTATTATAACAGAATACCACAAACTGGCTAA CTCACAGTTCTGGAGGCTGGGATGT TTTGGTCCCTTGATCCTCAATCTTAGACATCCCAGCCTCCAGAACTGTGAG

629 1223 TCAAGCGATCCTCACACCTC CACTTGAACTCAGGAGTTTGAGAC TAATTTTTGTATTTTTTATAGAAAC TAATTTTTGTATTTTTTATAGAAACGAGGTTTCGCCATGTTGCCCAGTCTG

630 1224 GGCCTCAGTGAAGGTCTCCT CCACTCAAGCCCTTGTGC TGCAAXGCTTCTGGATACACCWTCACC CAAGGCTTCTGGATACACCWTCACCGCTACTGTATGCACTRCCAGGTCCAT

631 1225 AAATAAGGGATTCCAGCAATCAA GAAATTGGAGTATGATTGAGAAATGC GTGGTTGGGATTAAGGAAAGGAGGA CCATAAACCCAGAATCTAATTATGAGTCCTCCTTTCCTTAATCCCAACCAC

632 1226 TATCATGAGGCAGAACTGTGAGAT CAGGGTTTCACCTTCCCCT CCTTGGCTAGTTTGTCATTGAGATG ATGATGGGTTCCTGGCTCAGCTTTGCCATCTCAATGACAAACTAGCCAAGG

633 1227 AAAACTCCACATGATGTTTGCAG GTTTTCTTTCCTTGGAATGGTGA TCTGGAAAAGAAAGTAAGCACTGGT CCTGGTAGTTTCAGACTCCAACTGGGACCAGTGCTTACTTTCTTTTCCAGA

634 1228 TATCTTCAGAAGGAGTTGGCTGG CCGGCAGCTCTGTGAACA GTCTGTGAGAGCCAATGAACCCTTT GTCTGTGAGAGCCAATGAACCCTTTGGAAGTTCTCAGATGGCCAATGAGGA

635 1229 ATCCTCCCAAACACCTTTGC AAATTTGCTTTGATATCCCAAAGTG ATCAGTAGGCTCTTGTGAAGGAAAG ATCAGTAGGCTCTTGTGAAGGAAAGATGGATGGCACTTCCCAGATATACCA

636 1230 GCTTTCCCAAGAGTTGGAATTG TGGTATCGAATAATGAATTGTATTCAG ATTTGTATACTCATGCTGXACTACCTA TTGTATACTCATGCTGTACTACCTAGTACAATGGCCTGTTGAGCTGAATAC

637 1231 AGGAAGCTGAAGCACGAGAAT TTTTCTTTTTTTTAAGACAGGGTCTC CTTCAXCCAGAGAGGCAGAGGTTGTAG TCAACCAGAGAGGCAGAGGTTGTAGCGAGCTGAGATCACACCACTGCACTC

Row# REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

638 1232 TTTAAAAAGTGGCTTTCACCTCAT CTTTGGCAGAGAAATGTTAGGAAG TGTGAAGGCATGGGGAGAAATGTGA TGTGAAGGCATGGGGAGAAATGTGAGTTATAAATAGCCAGGAGAAACTATG

639 1233 TTGGACAGCAAAGGGAAAA GAGCTCTGAGAACTACAGACTTGACTT ACTTGACTTGCAAACACAGATTAGA TAAAAAGATGTTCTTTGCCCTCCTGCTCTAATCTGTGTTTGCAAGTCAAGT

640 1234 AGGGCTACTGCTGCTCCAG CCTGGCCCCATGACAGCT CCATGAACACCTGCACGACAGACAC ACATGCCTGTTGGAGTGGTGGCTCCGGTGTCTGTCGTGCAGGTGTTCATGG

641 1235 AGTATTGAGAAGAAGGCTGGCAT CATCCTTTGAACTGGCAAGG CTGGAATAGGAAXGTTTTTACTTACAA ασAATAGGAAAGTTTTTACTTACAAAACAACAGCCAAGTCAGTCTGGCATC

642 1236 AGTATTCTTAGTAAGGGCTCGCA AGCTGGCAGGAAGCGGGC GTCCCCGAATATGGCATTAGTACCC GTCCCCGAATATGGCATTAGTACCCGATACCCGGCTGAGCTCAGACTTCCA

643 1237 CACCCCACCCACTTCCTT ACTGAATTTATTATAGCCTTCATATTTCCTG GTGTGTAACAATTATTTGAAAGATT GTGTGTAACAATTATTTGAAAGATTGAGTGTAACATTTCAOGAAATATGAA

644 1238 GCCATGTTTCACACTTTCTGTAATT ATATCCACCATGAGGACCACAG GGAATGTTXATTTGTCTTGTATTAGTG AATGTTAATTTGTCTTGTATTAGTGGAGGCTGCAATCAGTGATATTACTGT

645 1239 ACTGACACGTACCGCATTATAGTC CAATATGGAACCCGAACCAC GCATGTTTACAAAACCCCCAGGCGA GCATGTTTACAAAACCCCCAGGCGACACATGTGCACATTAAAGCTGAGACA

646 1240 AACATGGTGAAACCCCGTC CTCTGCCTCCTGAGTTCAAGC AAAATACAAAAATTATCCAGGCGTG AAAATACAAAAATTATCCAGGCGTGGTGTGGGTGCCCGTAATCTCAGCTAC

647 1241 GAGCATCTGCCCCAAACC TGTAGAACCATTTTGGTGTCTTGG CAATTXGTTCTTATACCAGXGCTATGT AAATATCGCTTTCTAGAAGAGAAGACACATAGCTCTGGTATAAGAACCAAT

648 1242 CTCCCCATCCTCTAGTTCTCTATTTT ACTACATTCTTATGCAAGTTGTAAAGGC TTGTAAAGGCTAAATCTCATAAAAT CTTGGTGTTATGGCCTATATTAAAAGATTTTATGAGATTTAGCCTTTACAA

649 1243 TTTTGTGATAAGTTAGAATTAGGTAAGAGAACA CAATGGTCTTCATTCAGGTGCT TTAAGCAAGATXCCAGGCAGAAGGAAC AAGCAAGATTCCAGGCAGAAGGAACGTGGCAAGAGGGAGCAATAAAGGCCA

650 1244 CCTAACACCACCACTGAGAGC CAAATACAATGATAGGAGCAAATGTCT GTAAAAAGCACTCTAAGAACCCAGA TCACCAATTCCTCACCCGGCTCCGCATCTGGGTTCTTAGAGTGCTTTTTAC

651 1246 CCCGCTCCAACCTCAGAG CATTATTTTTTGTTTGCCAAGACC GCAAATTCCCCGACATCAGAGAAGC GCAAATTCCCCGACATCAGAGAAGCAGCCCTGGGCCACTAAACGGAAACCT

652 1247 CAAACTCCTTTTAATAAACTCTCTGCC ACTCAGCAACTTTTTGTCTCCATC CTATTTACTCAAACAGGAAGAAAAC ATACATTGAAAAAATAAATGCTCTCCGTTTTCTTCCTGTTTGAGTAAATAG

653 1248 GTACCTTACACTTGGCTGTATTCCC GTGGAAGGCATTTACATCTTTGG TCAGTATCTTCCCXAACXTGGTTCCCA ACTTTCTGAACCTGAACAGGAAACTGTGGGAACCATGTTGGGGAAGATACT

654 1251 CTGTTCTCTAGCCTGGGTGACA TTCATAGTTGTTTCTGATAGCATTTCAG ACATATAAAXTTAAAAGATGAAATT AATCTCTCAATTAAAAATGGATCCTGAATTTCATCTTTTAATTTTATATGT

655 1252 CTTATTTGTTCCCAATTCCGG GACATCTAATTAAATTGATCATCTACAGTGAT AGTAAATTACAAGTATGAXCTAAGTTT TAAATTACAAGTATGAGCTAAGTTTACTTTGCCAGTAGTGTTTGGCAAACA

656 1253 GGCTGAACTTAAACCATTTTACTCAG AGGCAATCAGCACTCAAGAGG GTGGTTTGGTTTAAAACXAGGAAAAAA TACAGTGCAACTCATAAGCAAATGAGTTTTTTCCTAGTTTTAAACCAAACC

657 1254 TGCCTAATGTACTACACATGATCGG ACCTGGCACTCATGTCTCCC AAGGACTGTGACCTTTGTGAAAAAT CGAAAAACTATTACAAATAAGTAACGATTTTTCACAAAGGTCACAGTCCTT

658 1255 TCCCAAATCATGACATTTTCTTG ACATTTCTCTCAGATGTCCTTTTAGGTA CCTTTTAGGTATATACTXTGTTCACAT ACATTTTAATGAAAACATGATTAAAAATGTGAACACAGTATATACCTAAAA

659 1256 TGGGCTGTACCCATTTTATTTG TCTCGCTTCTCATAGAGCACG GGCAATCATTTAATTCXTGTTCATAGA CAATCATTTAATTCATGTTCATAGAGGGAATATTTTAAGAACGTATAGGGA

660 1257 TGACGGAAACTTCCATAGAAGAAA GCCTGGGACCACTGCATAT AGGGCTTTAAGAGCAGTGGTTGTTA AGGGCTTTAAGAGCAGTGGTTGTTAAGTTTTCATTTATGTAGGAGACAAAA

661 1258 ACCACTTCTCTCTGTACTTTTTCTTCATT CATGCAGGATGAATATACAGAGTACCT AAATGAGAGGCXGXGACCAGGCAACCC ATGAGAGGCTGGGACCAGGCAACCCCGTGCATCTGGTGGCTGGTGACTCAG

662 1259 TTGTCAGGGTATTTTCCGTAGAGA GTTTCTCGGCTAACACCCTC CTCCAAATTACCTATGTGTGATCTA CTCCAAATTACCTATGTGTGATCTAGTTATAAAGATTTGCAGGGAGCAGCT

663 1260 TTTAGTCCAAGGGAGTCCTGC GAACAATGCAACGCCCTG CAAACATGAATCTGGGXACGGAAAGTA TGACTACCGCCTTATTAGAGAGTCAGTACTTTCCGTACCCAGATTCATGTT

664 1261 AGAGATTTGATGTGGGACAGAC ACCAAGAAGCCAAAATATGAAATAAAC ATTTTATCCCATTTCATTTTGACAC ATTTTATCCCATTTCATTTTGACACCGGCTTTATTTCAGTTTTGGAGCAAA

665 1262 CTAACGAAATAATACCTTGATCACTTGAA TGAAAGTTATATTTGGAATCAATGAGG AATGAGGTGATATGGTTTTATGCTT CACATATACCTCATATACATACACACAAGCATAAAACCATATCACCTCATT

666 1263 AAGAAAAGTTTCCTATGGGAAGGAC CTGAGATTTGAGGCTACTGTAAATCC ATGAGTATTAGTGGTCTTTCTGTCT ATGAGTATTAGTGGTCTTTCTGTCTCCCTCAGGGCCACAAAATTCCCCTGG

667 1264 CATTCAAGATTTTTTCTGAAGTTATTTCC TTTTAAAGGAACAGTTTGAAAACACTG ACTGTTGTTGTACAATCTGTGAAGG ACCATAGTTGTGGGCTCTGAAATATCCCTTCACAGATTGTACAACAACAGT

668 1265 CTTGAGTCCTCCTCCATATCCA TTTGTTTTAGAACATTTCGCCTC AATAACACATTTACATACACCGAAT AATAACACATTTACATACACCGAATGTATCTTCTGTTAGAAAGGTGGAGGC

669 1266 ATCATCATTAGTCAAGCTATTCAGAGG CATAAAAGCATTTGCTCCCTG GGTCACCAGGGAAACCAGGCGAGGA CTCCCACCCATCTTCCAGGTCCCTCATCCTCGCCTGGTTTGCCTGGTGACC

670 1267 GGGGAGACTTCTATTTTATGGAAGAA AGCTATCTTATGGTAGAATAGCCACTTTAA CATCCTCAGTGATTGGAAGATTATC CATCCTCAGTGATTGGAAGATTATCACAATGGAAAGGCCTTTTAAAGTGGC

671 1268 TTGCATTTTCTGATCCTTCATCC GAGAGAGGGGTGCCATTTACA AATGTTCTTCCCTCAGTCACCCAGA AATGTTCTTCCCTCAGTCACCCAGAGGATGTTTTCCTGAACATCATTCAGG

672 1269 ATGAAGTCTTATGAGAATTGCCTCTTATT CAATCCCCCATTCATCTCAG GTCAGGGTGAGTTGAGTTTGGGTAG CCCCAGTGCTGAGATACACAAACTTGCTACCCAAACTCAACTCACCCTGAC

673 1270 CCAAAAAGCAATTAGGCATATAAATAAC AGTGAGTCACCTGTAGACAGCGTAT TTGTTTAXATCCATTCTGCCAATTTGT TGTATTACACTCTTCAAATGAAAGACACAAATTGGCAGAATGGATTTAAAC

674 1271 TCTGAAGTCGAAGTACTCGCC CTAAAGTCCAGCTTCTCATGCTG GAGGTCCGTGCGCTGTGGTAGCAGG GAGGTCCGTGCGCTGTGGTAGCAGGGGGGAGCCCCGCCAGCCAAATGCCAG

675 1272 GTTTAGCAAACTTTGACAGGCAA TGAGCAACTCAAAAAGAGAAAAACA AAAAAAAAAAAGAAAAAAACCCTGA GTGAACTGATCTTATAGGAGAGATTGTCAGGGTTTTTTTCTTTTTTTTTTT

676 1273 ATGCAGAAATCCTCAGCAAAATATTA CCTTGCATCCCAGGAACA CAAAGCCXACTTAATTGTGTTGGATTA TCAAATCCAGCAGCACGTCAAAAAGCTAATCCAACACAATTAAGTGGGCTT

677 1274 CCAGTGATTCCACAGTACAAAAGAG ACAGAAATGCAATATTCGTTACAAATG TCTTCTTGTAATATAACATTTCTGA TCTTCTTGTAATATAACATTTCTGACTTTAAAGAGATAAAGTATCAACATT

678 1275 GAATTAGTCTCTGAGGGCATGAACTA TGCATAACTATTGTATGCCAGGC GGCACTGGATCAAGGAATACAAAGT TTTTATTCATCTTTATATCTTTATACACTTTGTATTCCTTGATCCAGTGCC

679 1276 CAGGAGGTCAATGCCGGG CAAACTTCTCCATCGCCCTT AGAGATCCAACCACTCAATCCTAAG CTGCATCCCACCTTGCTGATGAGGAGCTTAGGATTGAGTGGTTGGATCTCT

680 1277 ACATAGGGTATCTTATGTCCCAGGA TTGACACAAAAACATCTGAACTAAGAAC CTTTGGAATGXACAGGTACAGGATCTC TTGGAATGTACAGGTACAGGATCTCGGAATCTTAAGTTTTGGTTGTTCTTA

681 1278 TCTTTTACTGTGTGCACTGGTGA CTCAAAAAACAAGACAAAAATGCAC ACTGGCTGCTAGGAGAAAGTGTGGT ACTGGCTGCTAGGAGAAAGTGTGGTCCCATAAGCAAATATGTGCTTTACAT

682 1279 GAGAAGTTTTCTAATATCCTTTAAGTTGCTATC TTCATTTCCTCTCCAGAACCC ATTCTGATAAATGGATTGAACTATG ATTCTGATAAATGGATTGAACTATGCTTCTATAATAAAATAATTATATGGG

683 1280 AGTTTGGGAAGAACAAAAGCTCA AATTCAAGTTGAGATTTGGGTGG GGGTXGGGACACAGCCAAACCATATCA TGAATTGTTGTTTTGCACTTACTCCGTGATATGGTTTGGCTGTGTCCCCAC

684 1281 ACCCTGAAAATCACCCAAATC CCTGCCAAGACATACACCATC ATAGTTTCAAXTAGAGCAATAGTAATA AGTTTCAATTAGAGCAATAGTAATACTGATCATGTATCAAAGAAAAATGCC

685 1282 ACACCTGAGTAGACCTCAGAGCC GTTCAGTGAACATTAGAGGGCAG AACXACXAGATGCATTTGGCAGTGTAG CCACCAGATGCATTTGGCAGTGTAGGGGCCAAGGGAAAGCATTCTTTCTGC

686 1283 ACTCCAAGTATTCCTTTGCAGATTC CAAAGTGAAATATCCTCACAAAAAAAGTA GAGCGGAATGCACACATCACAAACA GAGCGGAATGCACACATCACAAACAGGTTTCTCAGAATGCTTGTCTACTTT

RθW# REF# Upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

687 1284 CACAATCAGGTCTGATTTTACCAAA TGCATTACACTAGTATGAGAGAAATACAGAA AGAATGTCTGTTTTGXAXTTCCATG AACTTAGAAATACTTCTAACTTAGGGCATGGAATTTCAAAACAGACATTCT

688 1285 TATGTGACTTGCCTGTATCTCACAG CATTTCTCTGTAAATTTCACTCTCCTC AATGCTCAGCCXTTTAGGAXTAGATAT CAAACCTGAGGTTCTTAACTACTATCATATCTAGTCCTAAAGGGCTGAGCA

689 1286 TGTATATGGTGAGAGAAGGAGTCTAGTCT ATGCATTGGAAAATAAGGTAGTCTCTT TAGTCTCTTAAAAAAAAAAAAAAAG CATGCAAATATTAAATTTTCCCAGTACTTTTTTTTTTTTTTTAAGAGACTA

690 1287 CACTTCCTGTGACACCCCC GTCATGAAGGTGGGTGGG GGTGTGAGCCCCCTTGCTGGAGAGT AAACCTCGGCCCTCCTATCTTAATACACTCTCCAGCAAGGGGGCTCACACC

691 1288 ATGAATGAATGAGCAGTAGGCAG CAGAATACCCCAGAGACTCAGACC GCCTGTGACXAGGTGGTTGGGGACGAG CTGTGACCAGGTGGTTGGGGACGAGCCCCCCTCAGATCAGCCCCAGGTCTG

692 1290 CCGCTCCTATTGCTCTCTAAAAA GGGATGATTTAAAAGAGATAGATAAGTATATGC AGTATATGCAGCAGTGTATAGACGC TTTCTATGGGCATGCAATATTAGATGGCGTCTATACACTGCTGCATATACT

693 1291 AATGGAGCCCACTATCACAAA CATCAGAAACCCAGAAACCTG CTCTCXCXCGTGAGAGGTCTGTGTGGG AAGGTCAGGAGGGCCTTTCCCTGGGGCCCACACAGACCTCTCACGGGGGAG

694 1292 AAATCTCATTTTGAATTATAGCTCCCA CATCGAGATCTTGTGAGATGTATTCA CTCATGATTCAGTTACCTCTCACCA AATCCCCATATGTCATGGGATAAAGCTGGTGAGAGGTAACTGAATCATGAG

695 1293 AAATCTCAGGTTCCCTCCCA CTAAATGCGGAAGACAGCAGAG AGAGAGCAAGGCTGGGCGGCACACG CTGAGTCCTGCACATCTCACTCCAGGCGTGTGCCGCCCAGCCTTGCTCTCT

696 1294 AGCTTATCCAATGACTATCTCTGAAAATAA GGGGCATATGTTATGAGGATTCT CACTAACCCTCTTATCTAXATGTGCCC CTAACCCTCTTATCTATATGTGCCCGGCACTTGCCACACAGACATGGCGAG

697 1295 ACAAGAATTATTCTGTCACATGATATGG TAATAATAATGTGTAATGTTTATCCTGACTCATC GAGAGCACGAAAGAACCACATGCCC GAGAGCACGAAAGAACCACATGCCCGGGCCCTCAGCATTAGATGAGTCAGG

698 1296 AATCAAAAAGAAAGGAGGAGAAAGAA CTTAAAATTTCTATGCCCTCTTGTCTC TCAGGAGAAGTTTTTCAGGGTGTTA TCAGGAGAAGTTTTTCAGGGTGTTAGAGGAAAGGGTGGGAGAACCATAGGG

699 1297 ACTTTGTTTTCTGCCAACTGCA GAAGCTGATGATTGAAAAACTCCTC AACACGTGAACCCACCTGACTTACT GAAGACAAAACAGATGAGGAACACACAGTAAGTCAGGTGGGTTCACGTGTT

700 1298 TATCATATATAATATCAACCGAAACAGACAG GATAGAGGTCCCTGACTCCAGAC ACCACAGTTAATGGGGGAGATAGAC AGCCAGCAGTCTAATTGCTCATATGCGTCTATCTCCCCCATTAACTGTGGT

701 1299 GGGAGAGAGGGAGGAAGACT AGCGTCTGTTTAAATAGCCTCGA GCCAGGAAGAACAGAGGGAGGAAGA GCCAGGAAGAACAGAGGGAGGAAGACCTGCCAGCAAGAACAGGGAGTCGAG

702 1300 TATCTACTCAGAAACCACGCACAA CTTGCAGGGACAGTGGCTT CTCTCGAGTCCATGTCCCGCCGCAG GGAGCACTGGGTCTTTGGAGTCCGGGCTGCGGCGGGACATGGACTCGAGAG

703 1301 TGTTCCAAGACATAAAGATCAGGAG GATGACAGAGGTGGTCATGGC TTCTAGATGGCTGTCAGGTGGTGGG AGAAAAATTCAATGTAACTAAAGCTGCCCACCACCTGACAGCCATCTAGAA

704 1302 TTGGTGTAGAAGAGAAGCAAGAGAG CATATCTCTACAGAAAACCCACATTG TTGGGTGGCCCAGGCACTAXCTGTCCT GGGTGGCCCAGGCACTAGCTGTCCTGTCAAGGGGCAGAGCTCTCTGCAATG

705 1303 AAGATCATTCTTCCACGCCTAAC GAATACGGTGGTCTCTGGAGTC AATTGCAGTAACTCGCCTTTGGGAG AATTGCAGTAACTCGCCTTTGGGAGGGAGAAAGGAAAAGGTAGGCAATTTA

706 1304 AGATGACCCCTGATGGTTAGCT CAAGGAATTTCCAATGCTGTTG AAAAAATAGTTAATCTGTTCAGTTC AGCCCTCATTGAGTGCATCCTGACACGAACTGAACAGATTAACTATTTTTT

707 1305 GCTTTTGTTGTTTATTGTTTAAGACATCA AGGTGAATCGATCTGTGTGGTG AAAATCAGTAACAAAAATAACATCA TTCATTGAAGCTGTCTCACAGCTACCTGATGTTATTTTTGTTACTGATTTT

708 1306 TGTAGCTGGGACTACAGGCAT GTAATTCCAGCACTTTAGGAGTCC GATGCCCAGCTAATTTCTCTTTTTT GATGCCCAGCTAATTTCTCTTTTTTAAAAAAAAAATTGAGATCGGGTCTTG

709 1307 TGGCCTAGCCTCTTCCTCTG AATTTAACAGCGTGAAAGAACAGATT AGAAXAGXTTCGATGCAGGCTATCTGT CCCTTCTTCCAGGAGCCAGCATGCCGACAGATAGCCTGCATCGAATCTGTT

710 1308 AGGTGGCTCTATCTCAGAGAGGA TCTGCTTGACTCTGAAAATCACATT ATCACATTCTTXGCTACTATGCTATAA GACTATAAAGGGTAGATAAATATTACTTATAGCATAGTAGCTAAGAATGTG

711 1309 GGGGTGACAGTATGTATGTAACCAT ACGGTTTAGTTTCAATCTTAGAACAGTT CTATGCCCATTCACAGATXCAGTAGAG ATGCCCATTCACAGATGCAGTAGAGAGAAGAATTTCTTAAAGACAACTGTT

712 1310 TCCTTCCCCAAGAGCAGC CAAGGTCACAAAGTGAGGAGC AGCXAGCAGCGGAXACTTGTCTATGTG GAATTCCCGAGGCTATGCTGGGATTCCACATAGACAAGTCTCCGCTGCTTG

713 1311 GCACAGCCAAGTCTGAAAGTTT TGTAGTTCTGAAGTGTGGGACTTTAAA CAACAAATTGGTXCAAAGATGAAATAT ACAAATTGGTTCAAAGATGAAATATGTATCAATATATCTAAGAACCCTTTA

714 1312 AAATCTCCAGACCAAGAAGAAGC AGAAAGAAAGAAAGAAAAAGAAATTGAGG TATAGACCTGATGGAGAGCACTACG TATAGACCTGATGGAGAGCACTACGCGGTTCACTTGAAGACCCTCAATTTC

715 1313 GTCAGACAATATGCAGCAGTGTAAC AGGAATGAGTGTACCATTCTTTATTCAC TGTAATTAATATCTCAAAAAGGATG TGTAATTAATATCTCAAAAAGGATGGGTGGGGGAATTATAGGTGAATAAAG

716 1314 GCACAATCTCCCCTCATTG GGCACCTGTAATCCCACCTAC TACTCGGGAGGCAGAGGCAGGAGAA AACCTTTGCCTCCAGAGTTCAAGTAGTTCTCCTGCCTCTGCCTCCCGAGTA

717 1315 AAATTACTCAACATCACTGACTTTTAAGATAC CATGTTAGCCAGGATGGTCTC GATCTCCTGACCTCGTGXTCCACCCAC TAATCCCACCCCTTTGGGAGGCCGGGGTGGGTGGATCACGAGGTCAGGAGA

718 1316 TGAGCAGGGGGGATGGGT AGGGACAGCACTGGACTTCC TTCCATCTGXAGAGCCCCCTCCCCTCA CCCACCTAGCAAGCAGGTGCTGAGAGTGAGGGGAGGGGGCTCTCCAGATGG

719 1317 TCCAGGGTCTGGGTCTGC CATCGCTAACCAGGCCCA CTTXTGCTCACCCAGCCACXCTCAGAG TTTCTGCTTTGGCCCACCCTCCAGGGTCTGGGTCTGCAGCTCTGA

720 1318 TTGAGGCCAGGAGTTTGTGA CAAGTAGCTAGGACTACAGATGTGTGC TAGCGXGACCCTTATCTCTACAAAAAA GCGAGACCCTTATCTCTACAAAAAACTTAAAAATTAGCCGGGCATGGTTGC

721 1319 TTGAGAGGGGCAAAGCATAAT CATGATTCAAGGGGGAAAGG AGGAACCTGCAGCAGTCTXTAATAAAC AGTTTGTGGCCAGAGGACAAGCTGTCGTTTATTAAAGACTGCTGCAGGTTC

722 1320 TTTTGTGACCAGGTTAAACAAAAAGTA ATTACAGGCGTGAGCCACAA CACCTGGCCTCCATTGTTCATTTAT ACCAAAAGCGTGATTCCTAAAAGTCGATAAATGAACAATGGAGGCCAGGTG

723 1321 CTGCGCCTTCCTCTGCTC CCTCCACCTGCTGAATACTAGTAGC CTTCAACCATTTCACTTCTTTCGTG CTTCAACCATTTCACTTCTTTCGTGGCCACCACTTGGCTGGGATGCTACTA

724 1322 TGTTATTCAGATAAAAGACTGAGTTATGCA CAAGGAGTTTATGATCCCATAACTGA GAATGAATGAATTTGCCGATGTCAG GTGGGGGAGAGTGCAAGCATGAAAGACTGACATCGGCAAATTCATTCATTC

725 1323 ATGGACAGCTATGACATGATTACG GTAGTCCCACCTATTCAAGAGATCAA GTACAATTTCAGGTTCTCTCTATCG CCTGCAGGCATGCAAGCTTGTTATTACGATAGAGAGAACCTGAAATTGTAC

726 1324 TTCAACATCCTCCAGGACAGC CATATCGAGCCCCTGTCCC ACAGTATCTGGTCCAAATGTCAACA ACAGTATCTGGTCCAAATGTCAACAGCGCCAAGAGGGACACACCCTGCCCT

727 1325 AATCTTCCTGCCTCAGCCTC GAAGGTCAAGATTGGAGGTTTG AGTGAGACTCTGTCCCTAAAAAAAA CACTACACCTGGCTAAGTTAAAAAAATTTTTTTTAGGGACAGAGTCTCACT

728 1326 AGATTTTGTTCTACCTATTCCTAAGATCTGT CTGTGAGGATTAAATAAGTGAATGCA ATTTTTTTTCCTGCGTGCCAGATAC ATTTTTTTTCCTGCGTGCCAGATACGGAAATAAAGACTTTCTGTGCATTCA

729 1327 TATAAAAGGCTTATTTCAGTTAGAGAGTGTTC CAGTCTCGGGGGCTGATC GGTAAGGGAATGATCTACAGGGTCT CGACCAGAATCCCATGACCAAGGGACAGACCCTGTAGATCATTCCCTTACC

730 1328 AACTCCCCTCCTGCTCAAAC CTGGGCTCCCATCTTCAAG AGAAGATCCTTAACAXTXATTCTGT CCTGCAGGCATGCAAGCTTGGTGGAAACAGAATTATTGTTAAGGATCTTCT

731 1331 ATGCTACTAATTCTTGGTCAAAACCC CTTTTATAAGTTATGGCTGAAGACAAAGG TGTAATCCTAAATAAAACTXCGGTCTC GTCGACCTGCAGGCATGCAAGCTTTAGAGACCGGAGTTTTATTTAGGATTA

732 1332 ATCATCGCAGGGGTGGGG CCTTACCTCTCTCACAATAGAATATCGTAC AGAATATCGTACGXCGTGXGCTGGCAA TAGAGTCGACCTGCAGGCTTGCAAGGTTGCCAGCGCACGGCGTACGATATT

733 1333 ATGACAGTTTGACATGATTACGAATTC CTCCACAATGCTGGTTCCTAG GGCAGGATTTGAACCCTAACTCTGA AGAGTCGACCTGCAGGCATGCAAGCCTCAGAGTTAGGGTTCAAATCCTGCC

734 1334 CCTAAGCCACCTTTGGCAT ATAAGCCGTCTCCTCCATTTACC TATACATTCTATTCTTAGTCATTTT TATACATTCTATTCTTAGTCATTTTCTCCTTGTTCACCTGAGTTTCCTTCT

735 1335 TGATGGGAGCTCTGTGTGG CTTCCGTCATGAACTTTGGC GGCXCXCAGTGAGCTTGATAAGGGAGG CCCCCAGTGAGCTTGATAAGGGAGGAGAGGCTGAGAGCTGGTTCCAGCCCG

Ro # REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

736 1337 TTCTTGCTGGGGAACCTCA GAAAGAAAGAAAGAAAATGAATTTTGG TTTGGCATAAAAACTTGGGTCTCAT TGTAAAAAGCATATTGGTGGGAAAGAATGAGACCCAAGTTTTTATGCCAAA

737 1338 TGAGGTCGGTGCTGGACG CGCCACCACCCCAGGAAC CGAGTACACGCGXCCAGTACACAGGGC AGTACACGCGCCCAGTACACAGGGCGTGAGCTCATTACACGCCCCAGTTCT

738 1339 TGTAAAATGATTATGATTACCACACTCAA GATCTCAAGTGTCCTACACATGCA ACACACACACACACACACACACGGT TAACACATTAATTACCTTACCTAGTGACCGTGTGTGTGTGTGTGTGTGTGT

739 1340 TGTAAAATGATTATGATTACCACACTCAA GATCTCAAGTGTCCTACACATGCA CACACACACACACACACACACACGG AACACATTAATTACCTTACCTAGTTACCGTGTGTGTGTGTGTGTGTGTGTG

740 1341 TGTAAAATGATTATGATTACCACACTCAA GATCTCAAGTGTCCTACACATGCA ACACACACACACACACACACACACG ACACATTAATTACCTTACCTAGTTAGCGTGTGTGTGTGTGTGTGTGTGTGT

741 1342 CATGCCCAGCCTCAACAT AGATGGAAGTAATGAAATTTGTGTATGTC ATTTGTGTATGTCAGTGATXACGGTAA AATGCGAGACTTAAATCTACTCATGGTTACCGTAATCACTGACATACACAA

742 1343 ATCCACGCCCTACAAAGGG CATAGAGAAGGCAGAACCGAGA CTTTCCTGTTTCATAAGTAGCAATC CTTTCCTGTTTCATAAGTAGCAATCCCTGTGGTAACAGCCCCTGTGGTGAT

743 1344 AACTCCTCTATCACACACCGATGT CAAAATATGGAAAAAGCTCTCAATTG GCATTGTCGACTGGTGTTGATTTAC TTTCGGCATGACACAAAGCATGAGCAGTAAATCAACACCAGTCGACAATGC

744 1345 ATGTAAAATGAATGGCAAGGCA CATTGCTGTACCTGGAAAATGG GGGCCCACAGTAGCATTCTXCACTACA TCTTCATCCCGACGGCAACATACGTGTGTAGTGCAGAATGCTACTGTGGGC

745 1346 TCATGGCAAAAGGCAAAGG TTGTTTAAAAGTGTGTAGCACCTTCC GGGAGTGAGGAGGXGTGAACACGGCAG GAGTGAGGAGGTGTGAACACGGCAGGAACAGGAGCAAGGTGCAGGAAGGTG

746 1347 CTTAAATGAGTATAGCTGATGAATCAATGA CCAAAATTGTCACAAGTCTTTGATT GACAACTTTGGGCAGTGGGTCTCAA GACAACTTTGGGCAGTGGGTCTCAAAGAATTCAGCTTCCTAGGCCTTCAGA

747 1348 TTCTGTGCCTATTGGGGTGA CAAACTGTCTTTTTCTCATTCCTTTG TTCCTTTGTCTCCTCTGGACTTCGT CAACCAACACCAGGTCGTGGGGGGCGACGAAGTCCAGAGGAGACAAAGGAA

748 1349 GTTTTTCAATATAGTTTGTTACCTATTCACAC TTTCAAGATGACAACAGAGACATTAAATAT CAATTATTTTTCCTTTTTCAAGTGA CAATTATTTTTCCTTTTTCAAGTGACACATTCAGCATAACCTGAAAGATTA

749 1350 TATCCATGATACTTGTTTGTGCTCTC GTACACCAGAGAAACAAATGCATG AGTAGGTCTCCTGTCATCTCAGCAC AGTAGGTCTCCTGTCATCTCAGCACGAGGTACCAGTGCCTTTGGTTCATGC

750 1351 ATATTTTCCAGGGAGTAATCTTTATATTTGG CATCTAAATCTGGAGCTACCCAGA TTACTCTCAAGGGATAGTXCAGCATGT ACTCTCAAGGGATAGTGCAGCATGTGCTTTTAAAATGCCCTTCAGGACTTG

751 1353 TTTTGAGATGGATTTTTCCTCTTG CAGGAGAATCACTTGAACCTGC ACCTGCGAGATGGCGGTTGXTGTGAGC GGCTGGAGTGCAATGGCAAAATCTCGGCTCACAGCAACCGCCATCTCGCAG

752 1354 TGTTTGTTCCCCCTCTGGTAG CTATTTCCATCTTCCTATCCCCAG GCTACGTGGTAGCCTXGAAAATCAACA TTTTATGACTACAGTAATTGCAGGGATGTTGATTTTCAAGGCTACCACGTA

753 1355 GGGATAGGAAGATGGAAATAGAGC GATTCCTCCATGAAAACAATCAGA TTATCACAACTCTGAAGGCTAACCA AACACTTCTCAGTTTATTGAAAGCCCTGGTTAGCCTTCAGAGTTGTGATAA

754 1356 CCCTCTACCAATTTCTATATAAGTCAGGA TGAGTATAATACCAGAAGTATGACTGTTGATC TCATAACAAAAACCCAAATCTCAGA TCATAACAAAAACCCAAATCTCAGAGAAAAATACAACAAAAACTTATTTTA

755 1357 CCAAAGTCGAGTATGTTGCCA TCCAATCTGGTTGGTAAATAGCC ACAAATAAAAAATGAGGCAXATAGGCA AAATAAAAAATGAGGCATATAGGCAGCTCTAGACAAGGTCTGGAATGGCTA

756 1360 CATCTTAAGTGGTACATTTAAGTATTAGCTCC AGCTAAGTAAGTGACACATCAAAAATATCA CTCAATGAAATATGTAATGTTTTAT CTCAATGAAATATGTAATGTTTTATAATTGTTCAATTTTTAAAAAAATTTC

757 1362 AACATCTTACAGCAGATACTCCTTACACA AGCAGGGAAACTTCCTGTCAG CCTCCTACAXTTAGAATTGGGGGAATT AGGCAATCCTCTTGTTATTTTCGAGAAATTCCCCCAATTCTAATTGTAGGA

758 1363 GTCTTTCTGCTTCAGCCTTCAG GATATTAACAATATCTTTAATGRCATGCCT ATTXCATATCTAATCAGTGTAACAACA TACATATCTAATCAGTGTAACAACAACAGCTACTTAAAGAAGGCATGYCAT

759 1364 TGAGCGGTGGGGCTGGAG GTTTCACCCATTCCAAAAGTCC TCAGAACAAGGGATGGAGGTCTCAG TCAGAACAAGGGATGGAGGTCTCAGAGTTATCTGGCTGGATATGAGAGTTG

760 1365 GCCCTGAAGACTAACACACAGC TGAGGTCTCARATCTGCCATC AAGTTAACCTCAATGCTCXTGTGAGAT GTTAACCTCAATGCTCATGTGAGATCAGTGCTATTTCCACTTTAAAAGGAA

761 1367 CAACACAGCATCAGCAGTTCA TCGAAGTTCCCTTTGCTTGTC CAATAATGTGCATTGTGTTGAGTTT AACAAAGGTAATATGTCTTAATATAAAAACTCAACACAATGCACATTATTG

762 1368 GATGTACTGCTGAATACAGTTCGC TGTCACTCAAAATTCCCAGGA AGTATTTGGTTGAGGATTTTTTTAT AGTATTTGGTTGAGGATTTTTTTATCTGTCTTCATCAGAGATATTGACCTG

763 1369 GCTATGACCCATGCCCCG TTTACCAATGTATATGTTTATGTGTGTTATGC CACACATAGATATATXCACAAGTATAT CACATAGATATATGCACAAGTATATGTATATATGTGTGCATAACACACATA

764 1370 AGAGGGGAATATACTGTCTCTCAGTAAGT TAGAATTGATTTTTGTGTACGATGTAATGTA GAGTCGTTTCTTTTTTTMCCCCCCT ATACTGGGACATCTGGATATGCATACAGGGGGGKAAAAAAAGAAACGACTC

765 1371 AGAGGGGAATATACTGTCTCTCAGTAAGT TAGAATTGATTTTTGTGTACGATGTAATGTA TAATGTAGGAGTCGTTTCTTTTTTT ACATCTGGATATGCATAYAGGGGGGGAAAAAAAGAAACGACTCCTACATTA

766 1372 TGCCAGTCCTAAAATTCCCA GTGACCAGGGTTGTTGGC AATTAGTTCATTTTTAXAGTTTCTATA TTAGTTCATTTTTATAGTTTCTATAGAAATAATTTTTCCTATAAAAAACAA

767 1373 CATGCCACTGTACTCCAGCC ACAGGACATACAGAATTACTTTCAAATGA AGTATGTATGTAGGTATGTATGTAT CAAGACTCTGTCTCATAAATACATAAATACATACATACCTACATACATACT

768 1374 GAAAAAGATGGCAAGTACACACAAT GGTCTAATTTCATTTTCTCTGCATGT CATGTGAATXTTCATTTTCCTAACACT GTGAATGGACAGCCTCTTCAATAAACAGTGTTAGGAAAATGAACATTCACA

769 1376 CTCTTGGCATCATTATGGGC AACCTCTAAAACATGCTACAGGTAAACTATTA AAAAGAATGGAAAATCAATCAGAGG AAAAGAATGGAAAATCAATCAGAGGGCCTGGGCATTATAATAGTTTACCTG

770 1377 ACATGTCTCTATCTCAGCGTTCCT CTTTAGGAAGGTCACAATTCAGGC TCCCAACACTCATCAAGCCCATTAT TCCCAACACTCATCAAGCCCATTATATTTTCCCATGTGGCCACAATCTGTT

771 1378 AACTTTTCTGTCTAGCTAAAGGATTGTAAAC GATTGGTCCATTTACAAACCCTTAG TTGTAAACACACCAATCAGCACTCT TTGTAAACACACCAATCAGCACTCTGTAAAAACGCACCAATCAGTGCTCTG

772 1379 CTCAGTGGAATTGTGTCAACAGG AATTTTTTTTGGAAAAAAGGTCTCTG GGTCTCTGCTTTTTCTATTTTGGAT GACTGAAAAAAATTMTCAGAAACAGGATCCAAAATAGAAAAAGCAGAGACC

773 1380 CTCACACCTGTAATCTTGGCA CTTGATATGTTGCCCAGGCT CTGGTCTCAAACTCCTGGGCTCAAG TTGGGGCGGCTGAGGCAGGAGGATTGCTTGAGCCCAGGAGTTTGAGACCAG

774 1382 CATTGAATGTCTACCATATGCCTG TCATTTCATGTGTGTAAATCATGTTATG AGAAGACCTATGCCCTTTACTCAAA AGAAGACCTATGCCCTTTACTCAAAGAACTTATCTTATTGGCATAACATGA

775 1383 TTGTCTGCAAAGACTGTGAGAGAT TGGCCTTCCTCAGAGATGTG GCCAATTTACTTTCTTCTTTTGAAT ATACACACAACAGAAAAATATGACCCATTCAAAAGAAGAAAGTAAATTGGC

776 1384 AATGGCTTGATCAGAGTTCACTG AAAAATAAAAAAATTAGCTGGACATGG CTCCCGGGCTGAAGTGCTCCTCTCA CTCCCGGGCTGAAGTGCTCCTCTCACGTCACCCTCCTGAGTAGCTGGGACT

777 1385 AAGCCATTAAAGCTGAGAAGAGTTAGT AATTTGACCGATAAAAAGACACTCAG ATTGGTTAAGTTCACGCACCTAGAG ATTGGTTAAGTTCACGCACCTAGAGCGATGGAGCCAGGAACTGTCCTGAGT

778 1386 AATAACATTACGAGTGTTGGCAGC CATTTCTCCCAAGAGCAGGAA TGGGAGCAGAGCAACCTTGTGACGA CCTGCTCAGAGGGAGGAAGGTGGGACTCGTCACAAGGTTGCTCTGCTCCCA

779 1387 AGGCGGAGGTTGCAGTAAG CTTAATCAGATTGCCTGGTGC GGGTAGGGCCCGGAXACAGTATTTTTT CTCAAACAAAATAAAAATAAAATTTGAAAAAATACTGTATCCGGGCCCTAC

780 1388 CTCATAGAGAAATAGAACACACTCAGCT GGAATCCTAGAACATCACTACATAATTTCT TATXTTTAAATTACAAAAXCTACTCCC CTTACCTCTGAGGAGGGGAGCAGAACGGGAGTAGCTTTTGTAATTTAAAAA

781 1389 TTTTTAGCTCCAGATGACATTAAAAACT GTTAGCCAGGATGGTCTCGAT TCGXTCTCCTGACCTCGTGXTCCGTCC ATCCCGGCACTTTGGGAGGCCGAGGCGGACGGATCACGAGGTCAGGAGATC

782 1390 CCACTGAATTGTACACTCATAAATGG ATTTTTTATCTTGTGTTTATACATGTGTGTG AXAATGGXAATTTGGTTTTATATTTGC AATGGCAATTTGGTTTTATATTTGCGTATATATTTATACAGCTTTTTGTTT

783 1391 ACTCGCAAAAATATAGGTACACATCG GGTACCTGTGTCGAAAATCAGTTG TGTAACAGAATAGAAAGCCCAGAAA TGTAACAGAATAGAAAGCCCAGAAACGGGCCTATGCATGTACAGTCAACTG

784 1392 CAAGAAAACACATTTCCTTATTCACG GTGGAAGGTGTATCTTAAATTGGTCA AATTGGTCAAATAAGACXAGATGGAAG TGCTCTCCCAACCATAAAATATAAGGCTTCCATCTAGTCTTATTTGACCAA

Row# REF# Upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

785 1393 CTAGCAGGCCCAAGAACCTC GCATTGATTACATAGGAATACCAGATTT P CAAGXCTGTAAGTACAATCTAAAGTCA AGACTGTAAGTACAATCTAAAGTCAGTCTTGGTTTGGCTTCCTGTCTCAAG

786 1396 TCCTCCATGCCCGACCAC CATGAGGGGACTTAGTCTCTGAG P TCXCATGTCTTCXGGACCTGAGTTTGG TCATGTCTTCAGGACCTGAGTTTGGGTTGTGGTGGCCATCCGGACCCCCTC

787 1397 CAGGTCCTCTGAAACCTGGAT AAATGTGACTAAGTCATTACAAATGTGACT GTGAXTGAGTCATTACAGTGGACCCTC AAAACGGATTCTTTTCAATGCACCCGGAGGGTCCACTGTAATGACTCAGTC

788 1398 AAATTTGAATCCAGCCATACCTG CTTTTTCTACCTTATGCCCCTCA P ATTXACTTTAXCTGTGGTGAATCTGTA TCACTTTACCTGTGGTGAATCTGTACAGGTCTGCAGCTACCTCAGTTCTTG

789 1399 TACTAGTCTCAGAAGTATTTGTGTTTCAAGAA TGTTGGCCCATACCTGCTAA P TGAATTGTATTTGGGATTATGGAAA TGAATTGTATTTGGGATTATGGAAAGYAATGTTTTAATAAGTAAGACATAT

790 1400 TTTCCATGTGTGTGCCTGAG GATTTCTTTCTACACAAGGTCACCTG P ACCCATCCCCATTGGCTCTTCTACA ACCCATCCCCATTGGCTCTTCTACAGGTGATGTGTCTACTGTACACCTACA

791 1401 CTGAAGATTTATATTGATATTTGATGGAAAG TGGGCTACAGATCAAGAGCTAGG GGAAGAAAATATACCTTTAGAGTTA ATTCTGGATGCATACAGGTTATATGGTAACTCTAAAGGTATATTTTCTTCC

792 1402 AATATATGTGTGTGTATGCAGTTTAAATTACC ACTGAATGGCCTCCTGTGG P GCCAAGAACAAGGTGGTTXCAGTTAAG CAAGAACAAGGTGGTTGCAGTTAAGGTGGTTAAAGCAGGTCTGTGGTGATC

793 1403 GTTTTATAGGATTTGGGTAGATAAAGG CTTCTCCTGGCCCATCCT TGTGACCCCCACTCCTGCCCACCAG AAATTACAGTCAAAGGGGTTTGTTCCCTGGTGGGCAGGAGTGGGGGTCACA

794 1404 TAGGCTCCTGCACTTAGACTCCT CAGTGTCAGATGAGCAGCAGG TGGATGTTGTGCAGAGTGGAGAATG TCATGCTGCATCAGCTCTTGGCCCCACATTCTCCACTCTGCACAACATCCA

795 1405 ACCGGGAGGTGAAGGCCG CAGKCTGGGGAAGCTGGG P AGCCCCGCTCCCGTCCTTTGATTCT AGCCCCGCTCCCGTCCTTTGATTCTGTCCCAGCACACGGCACCGGCAGAGC

796 1406 TTTGATTCTKTCCCAGCACAC GGCCTGCCATTCTTCTGTG GTGCCGTCGGCXTCGGGAGGATGACAG ACCGGCAGAGCCCCAGCTTCCCCAGACTGTCATCCTCCCGAGGCCGACGGC

797 1407 GCTTGGCCCAGGAGTTCT TTTGACAGCAAAGACACTTCATTC AACCTAACCCATTATTTTGTTGGTC AACCTAACCCATTATTTTGTTGGTCGAAACAACAGCTACATCATTGAATGA

798 1408 ATGTAGCTGGTCAGTTCATGGTTTA GTTTGTGTGTCTGTACACACTCTTCTG TGAGTCATGGAACTCTGGGAGGGTC TGAGTCATGGAACTCTGGGAGGGTCGAATAAAAGCAAAAATTTCTCTCCTC

799 1409 AGAGATTTGGTGAGGGTTAACATGT CATGATCTTCCCATCTCAGCC CATTTAAACXAGAGGCAGTGGCTCACG TTTAAACCAGAGGCAGTGGCTCACGACCGTAATCCTAGCTACTTGGGAGGC

800 1410 AGTGTTTGTGTCCCTGGOTACTT GAAACATGTGCTGTGTCCACTC AGGGAGTGGTGATGACTCTXAACGAGC GGAGTGGTGATGACTCTTAACGAGCATGCTGCCTTCAAGCATCTGTTTAAC

801 1411 TAACCTCAATAATAACCATCACTTTAGG CATTTTTGGGCACCATCC TGGTGCCAAGCCXTCCAGGTGATTCTG CAATGTTTAAAAACTTGAGCATGCACCAGAATCACCTGGAGGGCTTGGCAC

802 1412 CTTTGGTGCTTCCAGCTCAG ACAAGCAAAAGAGAAAAAAAGCC CTGGTTTTCCATGACCTGAGAGACT CTGGTTTTCCATGACCTGAGAGACTGTGACCAAAGTTAACTTTTTATCTCT

803 1413 CAGTTATACCTTTTAAATCCTACTGGGC GCACTTGCCCCCAAATTT GAATXCACTGTTTCTGGAACATCAAAA ATCCACTGTTTCTGGAACATCAAAAGAGCAATGGCCTAATTAAGATAAATA

804 1414 CATATGCTAAGTGATCAATCGAAGTTAA AACTCTGGTCTTGGGACATGTTAA TGTTAATTAACATCTCTAAGCCTCA TAATACCATTTTACAAATGATAAAACTGAGGCTTAGAGATGTTAATTAACA

805 1415 AACAACGCTGCGCTGACA TTTGAAACTTCACTCGTTCCAGTT ACATCAGGAGGGGCCACGGTGAACA ACATCAGGAGGGGCCACGGTGAACACAGGAAATGGCTTTGGCAAATACTTG

806 1416 AAGTAGTAGGTGAGGTACTAATTATGGCAG AAGAGAAGCAATGACCCCTCA AACTCTTAAACTATTCAGTATCTCA AACTCTTAAACTATTCAGTATCTCAGTAACCACACTATAGTATATACTGTA

807 1417 CCATATCTGCAAGTTCTGAAGATCA TCACTTCTTAGTTTCTTTTCCACGC CGCTTATTTTCATTCATYTTGATAT GAACACACAGAATGTTTGTTGAATTCATATCAARATGAATGAAAATAAGCG

808 1418 GCTGACTTTGAGGTAGGCAAAG TCCTCTGCTTATTCCTTTCTGTTGT ATTGTATTAXAAAATTCTTATAGTGAC AAAAATGATCTGATACAGCTGAAAACGTCACTATAAGAATTTTATAATACA

809 1419 AATGCAGATTGTGGTCCTTATTACC ACTTTCCAGACTTGAAATTAGGTGACTA AGTCACATAAACATTCATCTTCAAA AATAACAATTACGATATTTATTAAAGTTTGAAGATGAATGTTTATGTGACT

810 1420 AGAAATCTAACTACAAGAGAAATTTGGTGA CCATCTCTTCCAGAGACCACC P CCTCAGGAGATGTAGAATGGTCACA CCTCAGGAGATGTAGAATGGTCACAGGCTGCATTTGTATGAGAGTCTGGGT

811 1421 CACCACAGAATGAATTTAGGTGAATAT TGGGGTTTATATTAGAATAGGTTTTAGGTT P CTGTTGTCCCAGGGCAGTAAAAAAG CTGTTGTCCCAGGGCAGTAAAAAAGGATACAGGCTACTGAACCTAAAACCT

812 1422 TTTAGGAGGACTCCAGTATTACCTGAT TGACAGGGTAGAGAGCCAACA ACAAGTAGGACTTTTCATTTATTTA GAGAAATCTGCAGAGAYTCTTAACCCTAAATAAATGAAAAGTCCTACTTGT

813 1423 TCAGAACAAACAGTTTAACAAAACCA CACCCTGGGCTCAGGACC P AGGGGGTTGTGGAGGCAGGGGGCTG AGGGGGTTGTGGAGGCAGGGGGCTGGGCAGCCTGGAGTGTAAGTGCCAGGG

814 1424 ATCTCTACAAAAAAGACAAATATTAGCTGG GCTCACGGCAGATTTGACC ACCTCCCGAATTCAAGTGXTCCTCCCC TCCCAGCTACTAGGGTGGCTGTGGTGGGGGAGGATCACTTGAATTCGGGAG

815 1425 TACTTATTTATTTCTATCCCAAGCTGTGA CAGTCTATTTTCCCGCCTTTTC CTTCCTTTGCCCCTCCCAACTTCCA GCGGCGATCTGCGACTGCGCRCAGGGTGGAAGTTGGGAGGGGCAAAGGAAG

816 1426 TCAAACTGCATTTTGGTTCACA CAGAACATTGCCAACAACTGG AGGAAATGGCGAAATAAAATAAAAT GTCATCCTTCCTTCTCTTTTTCAAAGATTTTATTTTATTTCGCCATTTCCT

817 1427 CTAAACATAGTTTGGTTCCTTTGTAAGTT TGTACAGATGTTTATCACAGCATTATGTC ACAGCATTATGTCXAAGTGGTTTTAGA TCCAGGTAATGGATGAATGGATTGCCTCTAAAACCACTTAGACATAATGCT

818 1428 TAGCACAGCATCAGTACAGCACTAA TCAAAATTAATGCAAAAGTGAACCA P TTACCTCAGATATTGTTATTTTGCT TTACCTCAGATATTGTTATTTTGCTCACATTATTTTGAACGTTATGGTTCA

819 1429 GCAGTAGTGGTTATGTAGCTTTTAATACTATTC TCTTTGTGCCATTTCTTTTTTACC ATATGCCTTTCTTTTTAGGGACTTA TTGCCCMTGGCTCRTTTTAAGATAAGTAAGTCCCTAAAAAGAAAGGCATAT

820 1430 TTTCCTCTCTTTAATGTGATTTCAGC ATAACTCTTCAGAAAATAACAGAAAATGTGA GTGAACXGTCAGTTAATACAACAGCCA TTTCCCAATCTCTTGCTTATGGCTACTGGCTGTTGTATTAACTGACAGTTC

821 1431 ACGAAGATCTCAAAATACTTCAAAACG CTTCAGGTGATGCTCCCG TTGGCCTCXCACAGTGCTGGGATTACA GTCGGCTAGGCACGGTGGCTCATGCCTGTAATCCCAGCACTGTGGGAGGCC

822 1433 AAAGCGAGAGGAGGAGTGG GCAAGAATTGAGGTTGCTGC GCTGCTAGTAACCCAGATAACCGTA CTTCTTAGAGAGAGGTTGGGGTGCAGTACGGTTATCTGGGTTACTAGCAGC

823 1434 ATCTTCTGAGCCCGGAAGTTT CCTTAGTTTCCTTAGCTGTCTTTTTATTTTA P CXACTXCATTTCAACCTGGGCAACAGA ACTGCATTTCAACCTGGGCAACAGAGTGAGACCCTGTCTCAAAAAAAATAA

824 1435 TCAACACACTCTATCCCAGCAC CAAAAATGTTTTCCCCACATAAAC GAGGGTCAGCAGGTTGGATAATGAG ACTTCCACGGTGTCACAATTAAACAACTCATTATCCAACCTGCTGACCCTC

825 1436 TACTAAAAATACAAAAGTTAGCCGGG CTCCGCCTCCCAGGTTCA AGCGATTCTCCTGCCTCXGCTTCCT GCAGKTGCCTGTAATCCCAGCTACTCAGGAAGCTGAGGCAGGAGAATCGCT

826 1437 GCCTTTATGCCAAATGTCTTAGACTA AGACCTCAAAAGCAAAATAATCAGG P TTCCAAAAGAAAATAGAGGAGTTAT TTCCAAAAGAAAATAGAGGAGTTATGGAGACCAATATTTAATAGAATAATG

827 1438 TGTACAAGACAAGGATGCCCA GGATACTCTTTATTTCTTTCTCTTGACTGG ATACTATXTTGAATAAGAGTAGTGAAA TTCCTCTAAGATCTTGTACAAGACAAGGATGCCCACTTTCACTACTC

828 1439 AAAATACTGAGCTGAGAGAGCTGTTC CAAAAAAGGTGCAGATTTTTACCTACT AGACCCAGGGTCAGGTGAAGTTCTT TTGACATCATAGTCCACAGTTTATGAAAGAACTTCACCTGACCCTGGGTCT

829 1440 CATGAGTGGATGCAGGGC CATGCAGGGTGTGAAATCCT P GAATAAGGAGCAGTGGAATAAAAAA GAATAAGGAGCAGTGGAATAAAAAAGCAAATGAGCGAGAGTTTATCAACAT

830 1441 AGAAAAGCTGTGATTTTAAAGTTTCCA CTCGGAGGCTGTCAAGATTATG ATGTTACACAGTCCCCACAAGTGCT GGTGAAGCCTAGATACAGAGGTCCACAGCACTTGTGGGGACTGTGTAACAT

831 1442 TAATCAAACAAAATAATCTGGCAGG GGCCCCTCTAACACACCTCT P CCCACAGGTGAATCATACAGAGTTT CCCACAGGTGAATCATACAGAGTTTAAAAAATGTTGGTATGTTTAAACCAG

832 1443 ATTATTAAAATCCCCCACGGAAG CATTAACAAGACACACAGGGTGG P CCCAGXGTTGCCTATCTCCTGACTCCA CAGAGTTGCCTATCTCCTGACTCCACTGCACTCCCATTTGCCTTCTTTCCC

833 1445 CTTTGGCATAGAGTAGGAGTCAAATATT TTAATGGTACACAAATAAGAAGTCAACAGT GAAGTCAACAGTACCAAAACCCATA CTTAGAGTAATGCTATTTCACAAACATATGGGTTTTGGTACTGTTGACTTC

Ro # REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

834 1446 GACAGACTCATCAGATTTAGGAAACC TGAGGCGAGGCTTGGTAA GCTAGTGACTGTTTTTTTTTGTTTG ACAGGCAAAAAAAAAAAAAAACCAAACAAACAAAAAAAAACAGTCACTAGC

835 1447 AAATAATAAAGCCAGGATCATGAGAA TCAAGAGAATCACGATTAGCCTTTAT CTATTGCTTGTATATCTCATTTCAT ACATTAGGAAAAAACAAGTGAATGGGATGAAATGAGATATACAAGCAATAG

836 1448 TTCAAAGCCCTCTTCCAGTTC CAAAGAATCAAGAATCAGTGTGGA P GGATGAAATGAGTGTCAGAGCAGGG GGATGAAATGAGTGTCAGAGCAGGGAATGGAAAGGAGAGTAGAAACTTTAT

837 1449 ATATTTGAAAGAATAAGCAGTATATGTCATTAGG TGTGCCATTTTTGCATTCCT GGATTCTGACCATTTCAATAGGTGC AAATTCAAAAAATGAAATACTATTACGCACCTATTGAAATGGTCAGAATCC

838 1450 AATTTTTTTAGAGAGATATCATAAGGAGCTC AAAATTCAACAGTTCTCAACCATTTC ACCATTTCCTTTAGCAATCGCCCAT TAGTTAATTTCCATCATAGTCTATGCATGGGCGATTGCTAAAGGAAATGGT

839 1451 TGCAGAGCTGGGAGAAAGAA GTAGTGGAGATAGTACCTGAACCCC P AGTGTGATAGAGTAGTAAGXGCATGGA TGTGATAGAGTAGTAAGTGCATGGAGTTTTACTTAAGTCAGAAATTGGGGT

840 1452 GTGCGATCTGGGCTCACT CATGCCTGTCATCCCAGC TACTGGGGAGGCTGAGGCAGGACAA AGTCTCTGCCTCCTGAGTTCAAGTGGTTGTCCTGCCTCAGCCTCCCCAGTA

841 1454 ATTGATATTCGCAAGGAAAACACA GTCACTGCTGCTGCCTTCC CCTTGGCTAATATGATTCAAAGAGA TTGCCCTTTCCCCTTGAATTTGGAGGTCTCTTTGAATCATATTAGCCAAOG

842 1455 TTCTAGAGAGGCAGGGCCTAG GACTCAAAGGAAGAGACTCAGGATC AGTGAGGACAGACCCAGAAACACAG TTCCTGGTTTGACCCCTTCCCACCCCCTGTGTTTCTGGGTCTGTCCTCACT

843 1456 CCACACAAGAACATCTACACTACGAT TGCTAACTATTGTATAACCTCTATAACCATGA TATAACCATGACTTGTGAGCAGTAA TTTTGCTCCTGTGATGGTATGTTAGCTTACTGCTCACAAGTCATGGTTATA

844 1457 CTAGCTCGGTACCCTGACCAC TGCTTTCTGCTGTAGGGTCTG P TTGAATAAGAXTTGCTCACAGAAAAAT GAATAAGATTTGCTCACAGAAAAATAAGCTTAAATCTACAATGAATGGCAG

845 1458 TTATTGAAGGCCCTGTGGAC CAGGATCTCCCAGGTCCATC CATCCXAATATGTGCTTTGTCCACTTT CCCAGTGATGCCCAAGGGCCTAGTCGAAAGTGGACAAAGCACATATTTGGA

846 1460 TAATGCCAGCTCCTCGGG GTATTTATTTAGAGACCGAGTCTCACTCA P GCGGGAGAATCGCTTGAACCAGGGA GCGGGAGAATCGCTTGAACCAGGGAGGCGGAGGCTGCAGGGAGCCGAGATC

847 1461 GTGGCAAAGCCTTCACAACTAG AGAAGTGAATGGGAGAAAAATAGAATATG P CCATCTGTAGACTGTCAAAXTATGTAT ATCTGTAGACTGTCAAAATATGTATGTATGTAATTTTGACGTATGAACCTG

848 1462 TTCAGTGAGTTGCGTGAATTC GGGCACCCTTATCTTTGACAC P TCTAGTGAATTATAAAATCTAAGGA TCTAGTGAATTATAAAATCTAAGGACGGATTTAGGAATCCCCTTAAATTTG

849 1463 ACCATTGGCAAGAGCTAAAAG GTAGCAGCGCCCAGTTTTC ATTGTAGTTCATTTGACCCTCCCCA TGGACACAAGCCGGATGGCCCGCGGATGGGGAGGGTCAAATGAACTACAAT

850 1464 ATTAACCTCAGCGATTGTTGCT CTATTGAGGAGAGGATGGGAGG CCAGGCAGGGCCACCAGGAAGAGCG ACACCTGGGTGAGTTTGGAGCGCTCGCGCTCTTCCTGGTGGCCCTGCCTGG

851 1466 TTGCAGAGTCTATGTCGATTCTAGAG GAATACTGTCGGACACAGCACTG P TGAGCAAGATTGTGACATAGGTAAA TGAGCAAGATTGTGACATAGGTAAAGTTGTGACGGGGGTGTTTTTTGTGCA

852 1467 AGGCCAAGCTAGTCCACCTC CATCCGGTAATTAGCGATGCTA TCCAAGGAACCAATCATXAAAGAACAC TGGCTTTCCTCTCTTTCTTCCAGCACGTGTTCTTTAATGATTGGTTCCTTG

853 1468 TCATTCCAAGGGTGATGGC AAAAAAGAAAGAAAGAAAGAAATTGGC P TGTAAGCAAGGGCAGCACTCTGGGT TGTAAGCAAGGGCAGCACTCTGGGTCCGACGGGGAGAGTGAGCGGGAGAGT

854 1469 AGGAATTCAAAATCTGGACAAAGAC TGTCATTTACTGCCCAGAATAACTG AACTGCCCTGXAGATTGCTTTTCTTCC GGCTCAGGAAAGAAAATTTCAGATGCGGAAGAAAAGCAATCTGCAGGGCAG

855 1470 CTTTAGCTTATACAGTGTGCATGAAGAA CAAGCACTTAAAAATTCTCTATTTTACATAAGG P AGCACAATGCAAATAGAAAACCGAA AGCACAATGCAAATAGAAAACCGAAATTTTATTTTCCTCACAAAATTATAT

856 1471 AGGTGCCCACTCATTCTGC GGGGAGCCAGCCTCTTCT P CCACATGGACCACAAGTGCTGTCTC CCACATGGACCACAAGTGCTGTCTCGGCTCAGTGTGGTTCCAGAGAACCTG

857 1472 TACTTGGATGGAGCATGTCAAG CTACTTTAGGCAGGGACCGAA P GTCCATCCATCCTATCAACAAATCT GTCCATCCATCCTATCAACAAATCTCTAGGAACTTCTACCATGAGCAAGGC

858 1473 TTATGAAAAACCCACAGCCAATAT CATGGATCACCTGTTCCTGG AATTCATTCTCCCATTTCTTAGGTT TGAAAACTGGCACAAGACAGGGATGCAACCTAAGAAATGGGAGAATGAATT

859 1474 TTGCCTATGGTTAGGGTTGTGA GTAGCCTTGTCTGTGGGCC P AAACCTCAGTGGTGCCAAAXATCAAAA ACCTCAGTGGTGCCAAAGATCAAAAAGGGTTATTTGGGAAAAGCATGCCCT

860 1475 CTTTGATGTTTATTGATATGACACCG AGTACCAGATTTTCAAGTTACATAAGTGAAC P TGTTTAAACAACGCAAGTATGTCCA TGTTTAAACAACGCAAGTATGTCCACGTGTTTCTTATAGGGTACACTTGAA

861 1476 ACGTCTTAAGCACCTTTCTGCA AAGTGACAGGATTCAGAAGCCA TATCAGAAAAATCCATCAGGXCTCAGC TCCCTACAACACTTTCTGTTGCTTGGGCTGAGCCCTGATGGATTTTTCTGA

862 1477 TGGAGGCCCAGAGGACGC GAAACTGAGTCTGGATAGGGGTAAG P AGGGACAXGATCTCTGTGATTTCCTAA GGACAGGATCTCTGTGATTTCCTAACGGGGACATCCCCAGGCTCCTCTTAC

863 1478 TGTGGTGAGCCAAGATAGCTACA CCTTCCCAATTAAAAGCAGCTAAT TTGATGTTCAGATTTTCCTTTTTTT AAGAGTGAAATTCTGTCTCAGAAAAAAAAAAAAGGAAAATCTGAACATCAA

864 1479 TTTAGCTTTGGGCAGTCGAC GGCCAGCAGCTTGTGGAT GTGCCCGGGATGCCTGTATCTATGC GTTTCTGTCTACTCCAACGTCCACGGGCATAGATACAGGCATCCCGGGCAC

865 1480 CTCATAAATTTCCATATTGTTTCTCTGTACT AGGAAAAACTGCCTCACCAAA P GTTCAAGTGCACAGATTTTTTTTTT GTTGAAGTGCACAGATTTTTTTTTTAAAAAAAACAGCTATTTGAATTTTTT

866 1481 TGATGGACAGGGCCATAAAG CTCACCTAACCCTGCCCC CTGACAGTATTTCTCCACCCGCCCT GTTTATGGTTTTTGTGATCAGCTACCAGGGCGGGTGGAGAAATACTGTCAG

867 1482 ATCCTGTTAACTCAGGAGCTGACC CTTTATTTTCCCACCTGTTAGACATTC ACAGXGATTTGGCCAGTTGTATTTCAC GGAAAACACTATCTAGTTCCATTACCGTGAAATACAACTGGCCAAATCACT

868 1483 TTTTTGAATGTTTTGGCCCA GGAAGCAGGAAAAAGGTCCC P ATGGCXGGCAGXTTCCAGCATAAGTCA GGCTGGCAGCTTCCAGCATAAGTCAAGCTCCCCATCCCCCAGGGGACATGG

869 1486 AATTCTAGCTCGGTACCCGG CATTTTACTTGAACACAGACCGAAG P TTAAATCTGAGTGXAACATCCGTAGAA AAATCTGAGTGGAACATCCGTAGAAGTAGCTCACAGACAATGCAGAAGTAG

870 1487 GCAGGTGTTCTGACTCAGCC CTTAAACCAGAACTGCCTAGCAAA P CTTTGATGXACACACTGCTGCTCATTC TTGATGCACACACTGCTGCTCATTCAATCTAGGGGCAAGTCTCATTTTGCT

871 1488 CCATTCACAGATGCAGTAGAGTGA AAATACTTGATAATTCTTGGACTTTCTCTTT P ACTGTTCTAAGATTGAAACTAAACC ACTGTTCTAAGATTGAAACTAAACCGTACTGGGTTTGAAAAGAGAAAGTCC

872 1489 CAACACAGTAACATATTCATGAGTTTTTAAA CAGGCTGAGACAACCCATCA P CACTTGGTGATCTCTGACAATGTTA CACTTGGTGATCTCTGACAATGTTACGTAGCCTGAACCTGGAGTTTTGGCT

873 1490 TCTCTTGCCTTAAATCTAGAGACAATCT CAGGTGGAACAGTGGAAGCA AGCGACAAGAATGTAGACAGGCTAC AGGGATAACTCTGAAGAACAGAATACGTAGCCTGTCTACATTCTTGTCGCT

874 1491 TGCACAACAGAAGGAAAGGTG CCTCTCTACACTTACCGTTTCTGC P TCATTCATCCACCTTTTCCTGTCTC TCATTCATCCACCTTTTCCTGTCTCCGCGTGTGCGATGCTCCCGCGTGTGG

875 1492 TCTTAGAAAGTGATCTTTGGGAATATAGG GAAATCTTCTATCTAGTCATGCCTCAGT GTCTTCTTCTCTAAATGCTTCCTCT TTGACATGTGGAAGAACTCTGTTTCGAGAGGAAGCATTTAGAGAAGAAGAC

876 1493 TTGTCTAAGTGCTCCTTGTAGAGAAG CAGAGACGCCTCGGCTAG P CAGXACTGACGAAGATGCGGACAAAGC GCACTGACGAAGATGCGGACAAAGCGGGGGAAGGACACCCCTGCCTTTGTG

877 1494 AAGGTGGTTAGCCTGGGG CCAGCCTTGCTGTGGGAG GTCTTTGGAGGGAATGAGTAGAGCC CTTCTCCAGAGGCAGATGAGACTTCCGGCTCTACTCATTCCCTCCAAAGAC

878 1496 CTCCCCAAGGTCGTGAATT ATGGTGGGTGTAGGTGCGT GTTTTCTCCCACCCCGTCCXGCTCTAG AGGCTGCCCTGGAGAACTGGAAGCCGCTAGAGCTGGACGGGGTGGGAGAAA

879 1497 ATACACACACATCTACCCCAAGGA CATCTGGGCTGATGGTGC P GACCAGATAAACACAXGTXGCCATCTT CCAGATAAACACACGTGGCCATCTTGGACCATGGGGCAGCCAACAATGTGA

880 1498 GAGAGTTTTACTTCTTCCTTTTTGGTCTA AGATTCTGGTGGCAGAAAGACA GACAAAAACGTACATTCATTTATTC TGCCACTTACATTTCTATGTATGTACGAATAAATGAATGTACGTTTTTGTC

881 1499 AAGAGTATGTTGAGACAGCAATAAGAG TCCTGATCTAGGTTCTGGGATC CCTAACTTCTCTCAGCCCCGTCTTC ATAGATGCACCCACCTGGCAGTCGACGAAGACGGGGCTGAGAGAAGTTAGG

882 1500 TGGAGGAAGAGGTATTTAAGGTTTG CCAGGTGTCAAAAACCTATACTATAATCTC P TAAGATCATATAGGGGGCTGGACGC TAAGATCATATAGGGGGCTGGACGCGAAGATCTGAACCTGAGATTATAGTA

Row# REF# upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

883 1501 CGTGGTGGCTCACACCTG CTAATTTTTGTATTTTTAGTAGAGACGGGG ACTCCTGACCTCAAGTGATACACCC ATCCCAGCACTTTGTGAGGCCGAGGCGGGTGTATCACTTGAGGTCAGGAGT

884 1502 AAAGCAAAAAACAACAAACGATC CAGAGGGCTTTGGATGCA CAGAAAGGTCAAXTGCCTCACTCAAGG GAAAGGTCAAGTGCCTCACTCAAGGCCACACAGCTAGCACTCAGGTCTGGC

885 1503 GGTTGAAACAAATCAAAACAAAAAAG ACCGTAACTGGGAGTCACCTG CAATGTTACAAGCTCAAGCATGGTA CTCCTTGGAGCTGTCCTAAATTCATATACCATGCTTGAGCTTGTAACATTG

886 1504 CCTGCCTCAGACAACACAGC GTGGCTTGGGGATTTGTG GGCCACCGGCCACAXCTCACCCAGC GACAGACGAACATCTGTTAAAACTCCGCTGGGTGAGCTGTGGCCGGTGGCC

887 1505 ATTAGGACTTATCCCTGTGCCAA CAAATAATATAAGAGCTCCTGAGTGGG ATATCTCTAGAGCACATTTCAAAGT GCTACAACTTTGTTGCTTCGAGTTTGACTTTGAAATGTGCTCTAGAGATAT

888 1506 GCTGAGTGTATTTGTATGGGTCTATTTC TCAGGTGACTGACACTATTCAATTTC TAAAACAGTGTAGTXTTGGAGAAATAA TTCTGTTTTAGTGATCTATTCGTCTATTATTTCTCCAATACTACACTGTTT

889 1507 GGAGTCTTTCCTGTTGGTCAAGT AGTTGAGGCTTGATAGGTATTTATTGAAT TCTGGGTTTGACTACTCATTCATTC TCTGGGTTTGACTACTCATTCATTCGTTTGATCTTTCATTCATTCAATAAA

890 1508 CCTTCGGAACCTTTCAACACA TGAGCTAGAAAAGTAAATGTGTCAGTCC GTCAGTCCTTGGTAGCXAAGTTTGTAC AGAGGATTTGGATAAAATTTATAAACGTACAAACTTTGCTACCAAGGACTG

891 1509 CCCAAGGTGCTCTAAGGCA GATAATGTGCTTTCAGGAAAAACAA CTTGXGCAAGTCAGTAACCCTGAGTGT GCCCCGTTTTACAGAGGAGGCAATTGACACTCAGGGTTACTGACTTGCCCA

892 1510 CAATGGATTTCAGGCTTCTTAGG TCTGAGTTTGGCAAAGAAAGTACC AGAGCTTTCATCATGGCAAGACATC AGAGCTTTCATCATGGCAAGACATCGACTCTTTTGAGGAAATTCCAAAGGT

893 1511 CCAGTGGCTCATCTCGTGG GTCTGATTTCTGGTGTTGGGG GACAAGGTTTCTAGAGGTAATCACT CAGCGGCTCATTCTAAATGTAATGTGAGTGATTACCTCTAGAAACCTTGTC

894 1512 GCAGTCTGAGAGATATGGATGACATAT AGTTAGGTTCATTTGTTTCATTTTCCT TTCTTTTTGACTTTTTATCATTGTA GACTCGGGAATCATGTAAGTTTCTTCTACAATGATAAAAAGTCAAAAAGAA

895 1513 ATTCAAGCACGAGAAGCCC CAATAGGTGAAGAGGGCCTCC TGAAGGTCATACAACCAGAACCCAC TGGGCACTGGGAGGAAGCTGCAACACGTGGGTTCTGGTTGTATGACCTTCA

896 1514 TTTATTCCCATCTTCTTATTCATAACTCG ATTGGAGCCAGGAGAGCAC ACAGAGAGAGAACCTAATCCXGCACGG GCCGGACGATGATGCCGCTGCTAGAACCGTGCGGGATTAGGTTCTCTCTCT

897 1515 ATCAACAACATCCCCTCCTCA GTACAGATGGGGGGTCATGA CCCCGTCACCCACCTCACTGTCCTC CCCCGTCACCCACCTCACTGTCCTCCTCATCCCTATTATCTGGGAGGTCCT

898 1516 GAGTAGTAAGGAGTTTAGAAGCGTACAAAC CTGACCCTCGATTGGTTTCC GCTCTGTACTCACATTGGATTTTTG GCTCTGTACTCACATTGGATTTTTGAGAGACCTTGAGGGTGGGAGCTTTTG

899 1517 TTTTCCAGTCTGAAACATAAAGGAA GCCCACCTTGGCCTCCCA AAGTGCTGGGATTACAGGCGTGAGC AAAATGCTATTGGACCGGGCGCAGTGGCTCACGCCTGTAATCCCAGCACTT

900 1518 CAAATACGTGTTAATACGACCATGA CGCCTCCGCCTCCCAAAG AGTGCTGAAATTACAGXCGTGAGCCAC TAAAAGGCCCTTCCTGGCCGGGCGCGGTGGCTCACGCCTGTAATTTCAGCA

901 1519 TCCGTGGCAAAGACATAACTAAG GTTGCTGTGAAAAGAAAGTAAATGAGA AAATGAGACAGAGGAGCCCAGCACT CCCGCATGTGACCTCCCCCAGGGCGGAGTGCTGGGCTCCTCTGTCTCATTT

902 1520 GAAGGCTTCCCTGAGCCTG GTGCCACTTGTAAGCGTGAAC TGTCCGTCTCCTCTTCAGCAAAATG CCCTCATTTGTGCTGGTGAAATGTCGCATTTTGCTGAAGAGGAGACGGACA

903 1522 CTGTTTTCAGGATTATCCAGCAG TCCTGGGTCAAGAAAAGTCTG GTTCGAAAATGTGTCAGTAAGTGTG CGTTATTTAAGAAATCGTGATGGAAGCACACTTACTGACACATTTTCGAAC

904 1523 GCGCATCATTGCCCTTTC CTTACTTTCTCCATTTACCTTAGAGGGA AATCACTTACATTAACAAGAGACTC ATTACAGACTTCACATCAAGTTCAACGAGTCTCTTGTTAATGTAAGTGATT

905 1524 AATATTCACCAGCCCTGCC GGCCTGAGGGAGGCAAAG AGGCACTCTCTAXGTGGCAACATCTCA TCTTATGGGGTTAGAAAAAGCTATCCTGAGATGTTGCCACATAGAGAGTGC

906 1525 TAATCCTTTTCTGCCCCACTG CATGTTCTTTTTGTGATGATGGC CAAXATTCTATCATACAACATCTGCTC TGGCGTGACTTTGGCCAATTGAATGGGAGCAGATGTTGTATGATAGAATCT

907 1527 AAATACTGATTTTAATGGTTTGTGATGC CTTCCTGTTCAACCTGTACCATATG CCAGGATCAAAAACTCCACAGGTGA CCAGGATCAAAAACTCCACAGGTGAGCTGCATAGGCAGCCAAGATTTAGAA

908 1528 CGCACACTCGGGACCCAA GTACAGACGCCCCTGCTG CTCCCAGGGATXTCCCAGCGTGAGAGC CCCAGGGATCTCCCAGCGTGAGAGCAGGGACGGGACTGCGCCGTCACACCT

909 1529 TAAACAAAGATGCAATTTACTACCAGG CTCCCCTGTGCCCAAGGT CCAAGGGGGGGGGGAAAACTTCAAC CCAAGGGGGGGGGGAAAACTTCAACGGTTCTCAGAAGGGGCCTTTCCAACT

910 1530 AACCCACATCTATGACATGATTACG GTCTTTTGACACCTATACGTCTAAGGG ATGTAAGGGTTTTTTGACTTTCTAT ATGTAAGGGTTTTTTGACTTTCTATGTGCTGACTTCATAAGATGTCACTCC

911 1531 AGACTCCCTGGGGCTAATTT GAACTTGTTGATGTGTCTGTGGAA AACACATGAAGATCCCAAGGXTCAGCA GGTGTCACCACTGACTTCCTTGAGGGTGCTGAGCCTTGGGATCTTCATGTG

912 1532 GGAAAGAGATGGTGTGGGG TGAGCAAGGTCATTGTAATACAGAAG TAGACGAATAATAAAGGTAAAGGTT ATTATTACAGGAAAATTTTAACCGAGAACCTTTACCTTTATTATTCGTCTA

913 1533 TGACCCCATCTATGACATCATTAC CAGGAGGGAAAGAGATGAACATT CATTCATGGGGTCCTTACAGCAGAT AACTGTAAAAATAAGGACATTACTACATCTGCTGTAAGGACCCCATGAATG

914 1534 TGCTTGTTGGCATGAGCA CACAATCGCCAGGAACACA TGCACATAAAAGCCAAGACTCACTT TGCACATAAAAGCCAAGACTCACTTCCTTTGGGACAAGTCAGAAAAATCCT

915 1535 AGCTTGACTCTATTTTAGCACTATTTG CTGCTATGGCTCTGGCACTAG CTTGACTCTATTTTAGCACTATTTG CTTGACTCTATTTTAGCACTATTTGGGGTGGAAATGGCAACCCATCCTTCC

916 1536 TGAACAGCATGACATGATTACGA CAGTGAGCTGAGATCACCAGC TTCGAGCTCGGTACCCCTGCCTCAT TTCGAGCTCGGTACCCCTGCCTCATGAAGGCCTTTTCTTCTTCTTTTTTTT

917 1537 CTGTAGCCAAGCAGGGGTT GATTCATGATGGGAAAACATCAA TGCCATCTTCTGACCACCCCAAACA AGCAGCCTαCAGCTTCAACAAGGACGTGTTTGGGGTGGTCAGAAGATGGCA

918 1538 AGGGGGCGTCTGCTCTCA CTCCAGGAGCTGTGGGGA TGAAGGGACACCCTGAGCGGAAGTG GACCCTATAGAAGGCTCAGCTTCCCCCACTTCCGCTCAGGGTGTCCCTTCA

919 1539 TCTCAGGAGTCAAAGCGCAT CACACTGAGAACCTCGCTCTG TTACCTGTGTTAAAAGCAAGACTCT TTACCTGTGTTAAAAGCAAGACTCTGCATGCTTATTCTCACATCAGTCAOA

920 1541 TTCTTTAATTCCAGGAAACAATATTCC GTTTCCAGGTGACAGGCAAA ACCTGCTXAAGTTATTCAAACTAGCCA CTGCTGAAGTTATTCAAACTAGCCAATCCTAAGCCTGCTTACTTGGCTTGG

921 1542 CTGGCCTCATGACCTTTTTTAA CTCATGACATTTTTCCCCACAT ACTACCATTCACCAGATTATCCCCC GTGTGAGTTATCCCATTGTACATGGCGGGGGATAATCTGGTGAATGGTAGT

922 1543 AATGTAGGAGTCACCTCCTCCT CTTTCTGGGGATGGGGAG CTGTCCCTGTTTCCTCATCCCCACC CTGTCCCTGTTTCCTCATCCCCACCCCCTTCTCAGCCACCCACAACACAGC

923 1544 TGGGTCCCCTGAATTTATAATGA AGTGCCTCTCTCAATAGTTGGCT CACXGTATCCAACGCAAAGCAAGACTG ATAAATAATAATAGCTAATATTTATCCAGTCTTGCTTTGCGTTGGATACTG

924 1545 ATCTCTACTAAAAATACAAAAGTAGCCAGG CAATCTCGGCTCACCGCA CCTCCCGGGTTAGAGCGATTCTCCT AGTCCAGCTACTCAGGAGGCTGAGGGAGGAGAATCGCTCTAACCCGGGAGG

925 1546 ATATGGGTTTTCATTTTTAAGATTGGT CGTTGCCTCCAGGAGAAAG AAGGCGTGCTGGCGTGAGXCCACAGTC GGTCCAGGGAGCTGCCCGGCATTGCGGACTGTGGCCTCACGCCAGCACGCC

926 1547 TTTTAGTTTTAAAGCTGAGAAAGCCC TTGTCAAATGGGTAGGTTACTCAAA TACTCAAAATTXCATAGTGGGACTGAA CAGAGCATGCTGTTGGCCAGGGATAATTCAGTCCCACTATGAAATTTTGAG

927 1548 GATTTCTAGCCTCCAGAATTGTGA AATGTCTTTGTCCTTTTTGGGC AAATGTTTGTTGTTCAAXCTACATAGT ATGTTTGTTGTTCAAGCTACATAGTCGATAGAACGTTTGTTATAGTAGCCC

928 1549 GTGGTGGCTTCTGTCTTAGGTC GTAATGGAAAAGGCCCAGGA CTCTCTCTTCCAGTGATTTCTCATA CTCTCTCTTCCAGTGATTTCTCATACTTTGAGGGACACTCTGGCCTGCTTT

929 1550 TGTCTCAACTTTTCCATTTCTGC TGTCCACTCACCTGCTCCG CCGACXCCCAAACCGCTAAGGAGAATT AATCCAGGCACAGGCTGGACCCTCAGAATTCTCCTTAGCGGTTTGGGAGTC

930 1551 TCAGTGCACCCACATGCC CGAAGTGCTTGGTGAAGGG GTAGTGGGTGCTCTGGAAGTACCGC GTAGTGGGTGCTCTGGAAGTACCGCCAACATTGACCTGCATAAGGGTCTAC

931 1552 GAATTGGTCCTTTCACAGCATC TGTGGTGTTTATGTAGTATCCTTTTTCC CAGATCTAAACTTTTCXTACAAGGTTA GATCTAAACTTTTCCTACAAGGTTAGGTACCAAAGGAAGAGCATTAGAGGA

1 2 3 4

RθW# REF# Upper PCR primer Modified Lower PCR primer Modified GBA primer Flanking sequence

932 1553 CCCAGGAGAGACAGTCAGGA P TTAAACATTAACCAGCTCCTTCCTC AGCXCACAGAGGAGAAGCGGAGACTCA ACTGGGAGGGCCAGGAGAGGCCTACGTGAGTCTCCGCTTCTCCTCTGTGCG

933 1554 TGCTTCTGCCCCCACAGG CATGACAGTTTTCTGGAGGCC P TATCAGAGGAGXTGGCTTTTCACACCA TCAGAGGAGGTGGCTTTTCACACCAGGGCAATATTTTTAATCAAGAGTGTG

934 1555 TTGCCTCCCTGGGTGGGC P CACACTCCCATGCAGGAGT TGACAGAGCAGTGAGCCAAAACCCC TCAAAACATGAGCTTTTCCAGGGGTGGGGGTTTTGGCTCACTGCTCTGTCA

935 1556 AAGCCTCAACTATATACTGTGCTCTG GATTTCATTTCCATCTCTTTTCCTAG P GTGGATATATTGAATXCAAAATCAGGC GGATATATTGAATGCAAAATCAGGCGTGGCCCTTGCCCTCATAGAGCCTAG

While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

Claims

We claim :

1. An oligonucleotide comprising a nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, or complementary sequences thereof, wherein n=0 through 934.

2. The oligonucleotide of claim 1, wherein X, when present in said oligonucleotide, corresponds to a single-nucleotide-spacer selected from the group consisting of anucleosidic moieties, abasic moieties, non-naturally occurring nucleotide analogs, and non- Watson/Crick base moieties.

3. An oligonucleotide comprising a nucleic acid that hybridizes with a first portion of nucleic acid selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+4, or complementary sequences thereof, wherein said first portion to which said oligonucleotide hybridizes also hybridizes with a nucleic acid selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, and wherein said oligonucleotide does not hybridize with a nucleotide base on said SEQ ID NO:4n+4, or complementary sequences thereof, corresponding to the nucleotide immediately adjacent the 3' end of said SEQ ID NO:4n+3, wherein n=0 through 934 and n is the same value for both SEQ ID NO:4n+4 and SEQ ID NO:4n+3.

4. An oligonucleotide comprising a nucleic acid that hybridizes with a first portion of nucleic acid selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+4, or complementary sequences thereof, wherein said oligonucleotide does not hybridize with a second portion of said SEQ ID NO:4n+4, or complementary sequences thereof, wherein said second portion of said SEQ ID NO:4n+4, or complementary sequences thereof, hybridizes with a nucleic acid selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, and wherein said second portion of said SEQ ID NO:4n+4, or complementary sequences thereof, also includes a nucleotide base on said SEQ ID NO:4n+4, or its complement thereof, corresponding to the nucleotide immediately adjacent the 3' end of said SEQ ID NO:4n+3, wherein n=0 through 934 and n is the same value for both SEQ ID NO:4n+4 and SEQ ID NO:4n+3.

5. An oligonucleotide comprising a first portion of nucleic acid selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+4, or complementary sequences thereof, wherein said oligonucleotide excludes a second portion of said SEQ ID NO:4n+4, or complementary sequences thereof, wherein said second portion of said SEQ ID NO:4n+4, or complementary sequences thereof, hybridizes with a nucleic acid selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, and wherein said second portion of said SEQ ID NO:4n+4, or complementary sequences thereof, also includes a nucleotide base on said SEQ ID NO:4n+4, or its complement thereof, corresponding to the nucleotide immediately adjacent the 3' end of said SEQ ID NO:4n+3, wherein n=0 through 934 and n is the same value for both SEQ ID NO:4n+4 and SEQ ID NO:4n+3.

6. An oligonucleotide comprising a nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+l, or complementary sequences thereof, wherein n=0 through 934.

7. An oligonucleotide comprising a nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+2, or complementary sequences thereof, wherein n=0 through 934.

8. A pair of oligonucleotides comprising two nucleic acids selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+l and SEQ ID NO:4n+2, or complementary sequences thereof, wherein n=0 through 934, and wherein said primers correspond to two consecutive SEQ ID NOs having the same value for n.

9. A triplet of oligonucleotides comprising a nucleic acid sequence selected from the group of SEQ ID

NOs consisting of SEQ ID NO:4n+l, SEQ ID NO:4n+2 and SEQ ID NO:4n+3, or complementary sequences thereof, wherein n=0 through 934, and wherein said oligonucleotides correspond to three consecutive SEQ ID NOs having the same value for n.

10. A kit comprising at least one oligonucleotide of claims 1, 3, 4 or 5.

11. The kit of claim 10, further comprising two amplification primers.

12. The kit of claim 11, wherein said primers comprise a nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+l and SEQ ID NO: SEQ ID NO:4n+2, or complementary sequences thereof, wherein n=0 through 934, and wherein said three oligonucleotides correspond to three consecutive SEQ ID Nos having the same value for n.

13. A kit comprising an oligonucleotide, wherein said oligonucleotide comprises a nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, or complementary sequences thereof, wherein n=0 through 934.

14. The kit of claim 13, further comprising two amplification primers.

15. The kit of claim 14, wherein said primers comprise a nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+l and SEQ ID NO:4n+2, or complementary sequences thereof, wherein n=0 through 934, and wherein said three oligonucleotides correspond to three consecutive SEQ ID NOs having the same value for n.

16. A kit comprising the two oligonucleotides of claim 8.

17. A kit comprising the three oligonucleotides of claim 9.

18. A method of genotyping a nucleic acid sample comprising hybridizing the oligonucleotide of claims 1, 3, 4 or 5 to the nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+4, or complementary sequences thereof.

19. The method of claim 18, wherein said oligonucleotide is employed in a primer extension reaction .

20. The method of claim 19, wherein said primer extension reaction is a single-nucleotide primer extension .

21. A method of genotyping a nucleic acid sample comprising performing a primer extension reaction employing the oligonucleotide of claims 1, 3, 4 or 5.

22. The method of claim 18, wherein said primer extension reaction is a single-nucleotide primer extension .

23. A method of genotyping a nucleic acid sample comprising performing a primer extension reaction employing an oligonucleotide comprising a nucleic acid, or fragment thereof, selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, or complementary sequences thereof, wherein n=0 through 934.

24. The method of claim 23, wherein said primer extension reaction is a single-nucleotide primer extension.

25. The method of claim 23, wherein said fragment excludes from 1 up to 10 nucleotides from the 3' end of said SEQ ID NO:4n+3.

26. A fragment of SEQ ID NO:4n+3, wherein n=0 through 934, wherein said fragment excludes from 1 up to

10 nucleotides from the 3' end of said SEQ ID NO:4n+3.

27. A method of genotyping a nucleic acid sample comprising: a) amplifying a target nucleic acid sequence that hybridizes to an oligonucleotide selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, or complementary sequences thereof; and b) performing a single-nucleotide primer extension reaction employing an oligonucleotide comprising a nucleic acid selected from the group of SEQ ID NOs consisting of SEQ ID NO:4n+3, or complementary sequences thereof,

wherein n=0 through 934 and is the same value in both steps a and b.

28. A mixture of reagents comprising:

(a) 10 to 200 mM buffer, having a pH ranging from 6.0 to 9.0;

(b) 15 to 250 mM monovalent-cation salt;

(c) 0.5 to 25 mM divalent-cation salt; (d) 0 to 0.02% volume exclusion agent

(e) 0.25 to 2.5 mM of each of dATP, dCTP, dGTP and dTTP.

29. A method of amplifying a nucleic acid sample comprising the steps of:

(a) combining two amplification oligonucleotides with said nucleic acid sample; (b) adding to said nucleic acid a reagent comprising :

(i) 10 to 200 mM buffer, having a pH ranging from 6.0 to 9.0;

(ii) 15 to 250 mM monovalent-cation salt;

(iii) 0.5 to 25 mM divalent-cation salt;

(iv) 0 to 0.02% volume exclusion agent

(v) 0.25 to 2.5 mM of each of dATP, dCTP, dGTP and dTTP. (c) contacting said nucleic acid with a nucleic acid polymerase.

30. An oligonucleotide comprising a nucleic acid sequence selected from the group of SEQ ID NOs consisting of SEQ ID NOs: 1-3740, or complementary sequences thereof.