AU699613C - Method of and apparatus for diagnostic DNA testing - Google Patents

Description

METHOD OF AND APPARATUS FOR DIAGNOSTIC DNA TESTING

The present invention relates to the diagnostic testing of DNA using polymerase

chain reaction (PCR) amplification followed by electrophoretic separation of the resulting

fragments to detect possible gene variants of mutatioπal defects and the like; being more

particularly directed to new and improved multiplex PCR techniques in combination with

preferably two-dimensional electrophoretic separation in denaturing gradient gels.

This invention pertains particularly to tests for the presence of DNA mutations in patients with inherited diseases, including birth defects (e.g. cystic fibrosis) and genetic

predispositions to adult chronic diseases (e.g., cancer). More specifically, the invention

relates to the quick preparation, by a novel two-step polymerase chain reaction (PCR)

amplification, of gene fragments and their subsequent efficient and accurate examination

for mutations.

BACKGROUND OF INVENTION

Genes with mutatiαπai defects (gene variants or alleies) can be identified by DNA diagnostic testing. Gene variants can be transmitted from parents to children. Some gene variants have a very strong effect and are, by themselves, capable of causing disease. Examples are many mutational variants ofthe cystic fibrosis traπsmembrane conductance regulator (CFTR) gene that cause cystic fibrosis. Other gene variants act in combination with gene variants from other loci. Examples include many of the common (polygenic) diseases, like heart disease and cancer. It is possible to test for gene defects in an eariy stage; that is, in cells from the embryo (pre-natal testing), but also at a much later stage in young, adult or old individuals.

By DNA diagnostic testing, information is obtained about a disease, sometimes before it has become manifest. This greatiy facilitates management of the disease, e.g., prevention, treatment. For example, it is possible to test for the presence of particular gene variants in cancers or infectious disease agents in order to predict, e.g., the course of the disease, response to therapy. It is also possible to test individuals for carrying a

particular gene variant, which they wouid not like to see transmitted to their offspring

(carrier testing). Finally, it is possible to test individuals at any time (from pre-natal to late

age) for inherited gene-encoded predispositions to disease. An example from one end of

the spectrum is cystic fibrosis, for which pre-natal testing and carrier screening has

already become relatively common. The other end of the spectrum involves late-onset

diseases, like cancers and neurodegenerative diseases.

DNA diagnostic testing involves an analysis of the sequence integrity of individual genes. At present, this is costly since accurate testing requires sequencing, or decoding, of the gene, which is labour intensive. Thusfar no cost-effective universally applicable standardized system for DNA diagnostics has become available (for a review, see Cotton,

1993, Current methods of mutation detection, Mutat. Res. 285:125-144). In order to be cost-effective and widely accepted a DNA diagnostic system must be accurate (more than 95%), have a high throughput, and not be labour intensive.

GENERAL BACKGROUND OF ANALYSIS TECHNIQUE

It is initially in order briefly to review the general techniques involved in PCR amplification and in electrophoretic separation of fragments, and where the art has applied and is currently applying the same.

A sample of cells, such as derived from blood, is first chemically and physically treated to extract DNA strands carrying genes that occupy only about two percent of the total DNA material in a ceil genome, with the remainder of DNA material cluttering up the

background. While each ceil has two copies of each gene and such can be identified by

building blocks or basepairs identified by sequences of letters A, C, G and T as a letter

code, they constitute such a small part of the long DNA strands that they must be

amplified by making many copies of the same to permit their inspection. This is effected

by heat-separating or denaturing ofthe DNA strand pairs, mixing with appropriate primers

to bind or anneal to the beginning and the end of the gene fragments (e.g., gene exons or coding regions) to be investigated, later more fully discussed, and adding sufficient building blocks to generate copies of the gene exons. The successive repeating of this cycle of steps effects a cumulative copying of the exons to produce a purified and amplified quantity - a process generally referred to as the before mentioned polymerase chain reaction or PCR amplification - and more fully reviewed, for example, in Molecular Pathology, Heiπ and Siivermaπ, Carolina Academic Press, 1994, Chapter 2, Molecular Techniques and Their Automation in the Clinical Laboratory, pages 5-31 (Winn-Deeπ). At this stage, it is then in order to inspect or analyze the gene exons to determine if there are mutations from normalcy. This is generally done by electrophoretically separating the DNA purified fragments, preferably on the basis both of size and base-pair sequence, as more fully described by co-applicant Vijg and A.G. Uitteriinden in Two- dimensional DNA typing: A Parallel Approach to Genome Analysis, Ellis Horwood, 1994,

particularly at pages 33-40.

Electrophoresis has been used not only for DNA fragment separation but also for separating other substances than genes, such as, for example, for protein analysis, as described in Electrophoresis 1993, 14, 1091-1198. Machines are provided for one-

dimensional DNA separation by electrophoresis with fluorescent dye labeling, such as the

ABI Prism 377 DNA sequencer of Perkin Eimeπ and for two-dimensional DNA typing, as

described by co-applicant Vijg and E. Mullaart et al. in Nature, 365, 30 September, 1993,

Parallel genome analysis by two-dimensional DNA typing, pages 469-471 , describing

apparatus of Ingeπy B.V. of The Netherlands. The first dimension (say horizontal)

application of the electric field to an appropriate gel matrix (later discussed) into which the

purified DNA fragments have been introduced, causes separation of the fragments by size, larger particles moving slower than smaller particles. By applying the electric field in an orthogonal direction (vertically) with a chemicai gradient as of successively more concentrated urea/formamide disposed in the gel, or a temperature gradient established thereaiong, the DNA fragments will migrate (this time vertically) until they melt and are locked in position in the gel matrix at particular vertical sequence-determined locations. To prevent melting of the entire DNA fragments, the latter can be attached (before the electrophoresis process) to a number of only G's and Cs, which are more resistant to melting than A's and Ts. This so-called GC-clampiπg, effectively locks each fragment in position which is determined entirely by the sequence of the exαn-part of the fragments. Is this sequence changed at only one position, for example, by the substitution of an AT couple for a GC couple, it will melt later or eariier and hence become locked in a different vertical position than the normal reference fragment

Such two-dimensional gene scanning (TDGS) has promise for becoming a cost- effective and widely accepted DNA diagnostic system. In this system, as above described, a large number of DNA fragments obtained through polymerase chain reaction (PCR)

amplification from a given DNA sample are eiectrophoretically separated on the basis of

both size and basepair sequence. This system is highly accurate (i.e., 99%), since the

second dimension separation is based on denaturing gradient gel electrophoresis

(DGGΞ). Indeed, DGGΞ is the only system with such high accuracy (Sheffield et al.,

1993, The sensitivity of single-strand conformation polymorphism analysis for the

detection of single base substitutions, Genomics 16, 325-332; Grompe, 1993, The rapid detection of unknown mutations in nucleic acids, Nature Genet 5, 111-117; Guidberg et

al.,1993, Molecular analysis of phenyiketoπuria in Denmark: 99% of the mutations detected by denaturing gradient gel electrophoresis, Genomics 17, 141-146). Automatic instrumentation for TDGS is partly available and partly under development TDGS allows the detection of all possible mutations in DNA fragments obtained from one or more genes simultaneously at a high throughput and with a minimum of manual interference.

One major hindrance to the widespread application of TDGS in DNA diagnostics is the difficulty of amplifying many fragments simultaneously in the same reaction tube by PCR (multiplex PCR). In fact, it is often not even possible to find PCR primer sites that amplify the relevant gene fragments and simultaneously fulfil requirements for both PCR and denaturing gradient gel electrophoresis, i.e., optimal PCR reactions and optimal melting behaviour of the amplified fragments. The current procedure begins with amplifying regions of the target DNA, usually the protein-coding regions (exons) of a gene, by PCR. These amplification reactions are conducted separately, e.g., if 27 exons in a gene are being analyzed, then 27 separate PCR reactions must be conducted. In practice, it is usually possible to conduct a few PCR reactions together in one tube (e.g.,

Edwards and Gibbs, 1994, Multiplex PCR: advantages, development, and application,

PCR Methods and Applications 3, S65-S75).

Clearly, with a large number of individuals to be tested and when more than one

gene is tested simultaneously in the same TDGS test, the total number of pipetting steps and individual reactions to be carried out can become very high. This increases the labour intensity of the test, but makes it also more complicated with a higher chance of human

error. Indeed, in view of this complexity, even complete lab automation where all pipetting

steps are done automatically will not solve this problem. The problem of not being able to PCR-amplify multiple fragments simultaneously under identical reaction conditions in the same tube is an important technical hurdle for TDGS to meet criteria for clinical testing, i.e., laboratory user-friendliness. To reduce the number of PCR reactions via multiplexing, i.e., conducting several PCR amplifications in one reaction by employing multiple sets of primers, is a non-trivial development Current approaches for multiplexing are sometimes as simple as combining a few sets of primers for which reaction conditions have been determined separately. However, in most cases multiplex PCRs must be developed with careful consideration for the regions to be amplified, the relative sizes of the fragments, the dynamics of the primers, and the optimization of PCR experimental conditions to accommodate multiple fragments. A key problem is the positioning of the primers. For gene diagnosis one generally aims at amplifying the exon sequences, splice sites and regulatory regions. Primers for exon-amplifyiπg PCR reactions are ideally placed in iπtroπic sequences adjacent to the exons. This provides some margin for adjustment of fragment length or amplification

quality as well as information about mutations affecting splice sites. These are the first

limits to primer choice.

Then, primers should be positioned so that non-specific amplification at other sites

than the target sequences does not occur. Indeed, the human genome is 3 x 10⁹

basepairs long, which provides ample opportunity for fortuitous sequence homology

between the target and other non-target sequences. This problem is not typical for multiplexing, but can also occur when one wishes to amplify only one fragment at a time. This is the second limitation to primer choice. For multiplexing, primers should be selected so that their predicted hybridization kinetics are similar to those of other primers in the multiplex reaction. This is a third limitation to primer choice. These limitations to primer choice, with total genomic DNA as template, are the reasons why multiplex groups are usually small (typically less than 5 fragments). For optimal separation in TDGS there is a fourth formidable limit to primer choice.

TDGS requires DNA fragments of 100-600 bp on average. One of the two primers should be coupled to a GC-rich fragment to provide for a GC-ciamp as highest melting domain in the fragment to be generated by PCR. This is essential to guarantee the highest sensitivity to detect mutations in the second (denaturing gradient) dimension gel (Myers et al., 1985, Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis, Nucl. Acids Res. 13, 3131- 3145; Myers, et al., 1987, Detection and localization of siπgie base changes by denaturing gradient gel electrophoresis, Meth. Eπzymol., 155, 501-527). Then, primers should be

positioned in such a way that the target fragment comprises only one single domain, that

melts earlier (at lower urea forma ide concentration or temperature) than the GC-clamp

attached to it It turned out that optimal PCR conditions for both PCR (let alone

multiplexing) and optimal meitiπg profiles are difficult if not impossible, to realize (e.g., compare the RB DGGE design by Blaπquet et al., 1993, Identification of germiiπe mutations in the RB1 gene by deπaturant gradient gel electrophoresis and polymerase

chain reaction direct sequencing, Hum. Molec. Genet 2, 975-979, with our present

design). The present invention involves a combination of so-called loπg-PCR with short-

PCR. Recently, PCR amplification methods were developed allowing the amplification of large fragments (up to 40 kb) from the genomic DNA. We have taken advantage of this development by using loπg-PCR to first amplify all the coding regions of the target geπe(s) in the smallest number of fragments possible. Using these long amplicons as template we then PCR-amplify the small fragments, required for the TDGS, in a multiplex format in this way the target sequence is first amplified away from the contaminating genomic DNA, which allows to obtain the small PCR fragments under identical conditions from this pre-purifled template.

Using the above procedure, primer sets selected only on the basis of optimal meitiπg behaviour of the PCR amplicons, also exhibited optimal behaviour in the PCR and even allowed extensive multiplexing. In part this phenomenon can be ascribed to the pre- purificatioπ by the loπg-PCR. Indeed, the long-PCR greatiy increases the amounts of target sequence relative to other genomic DNA sequences, thereby greatly decreasing

complexity of the reaction and increasing its specificity.

Although the above described phenomenon can be explained by the reduction of

complexity through the preparation of a purified template, an exact explanation can not

be given. Indeed, the sheer magnitude of the effect is surprising and necessitates a

reevaluatioπ of the factors involved in PCR optimization. One thing is clear, however. The

present invention alone enables one to design and perform an efficient TDGS test because primers can now be selected on the basis of meitiπg profile alone and multiplexing is greatly facilitated. The invention is also generally applicable. Indeed, selection of primers in every

PCR-based diagnostic reaction is an important issue and many potential priming sites turn out to give poor results. Multiplexing then generates additional problems, which is the reason that it is not widely used. The long-PCR short-PCR two-step amplification system offers an immediate and simple solution to this problem.

OBJECTS OF INVENTION

An object of the invention, accordingly, is to provide a new and improved method and apparatus for diagnostic DNA testing that obviate the above described difficulties. A further object is to provide for novel detection of mutations in genes by a two- step multiplex polymerase chain reaction amplification using long and short multiplex PCR followed by two-dimensional electrophoretic separation of the fragments on the basis of both size and basepair sequence.

Other objects wiil be explained hereinafter and pointed out in connection with the

appended claims.

BRIEF DESCRIPTION OF THE RGURE3

The invention will now be described in connection with the accompanying drawings

in which:

RG. 1 illustrates the sequence of process steps for performing the invention. FIG. 2 shows the meitiπg curves for RB exon 12- with and without the GC-clamp

(i.e. retinal blastoma gene).

RG. 3 shows maps of the tumor suppressor gene RB indicating the positions of the PCR primers for the long- and short-PCR reactions.

RG. 4 shows a computer print with the predicted positions of the short-PCR fragments in a 2-D gel electrophoresis pattern with the indicated specifications.

RG. 5 shows the actual gel separation pattern indicating correspondence with the theoretically predicted pattern.

RG. 6 shows that with the loπg-PCR product for exons 18-23 as template all 6 of the short PCR fragments are obtained (lanes 7-12), whereas with total genomic DNA as template most products are missing (lanes 1-6). It also shows that only 5 ng total genomic DNA is sufficient as starting material for the loπg-PCR (lane 13), and that all short PCR products are obtained with the Iong-PCR products as template (lanes 20-24).

RG. 7 shows details of wiidtype (homozygous normal) fragments and several heterozygous mutants.

SUMMARY OF THE INVENTION

In summary, in an important aspect, the invention embraces a method of analyzing

predetermined gene exons derived from DNA, that comprises, adding primer pairs to

successive groups of the gene exons followed by effecting polymerase chain reaction amplifications thereof in a common tube, as a first step and relatively long multiplex polymerase chain reaction; adding further primer pairs to each of the gene exons and effecting polymerase chain reaction amplifications thereof in the common tube as a second step and short multiplex polymerase chain reaction; and electrophoretically separating the gene fragments.

Preferred and best mode techniques will now be described.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention, as before stated, involves the design of an accurate and efficient mutation detection test on the basis of a minimal number of two-step multiplex PCR reactions in combination with automatic two-dimensional separation ofthe fragments to detect ail possible mutations in the geπe(s) simultaneously.

Table 1 lists the different steps in the design of a TDGS test, with the RB (retinoblastoma) tumor suppressor gene as a model. Rrst, the gene sequences are retrieved from a database (e.g. Geπbaπk) and the target regions, i.e., exons, splice sites,

regulatory regions, are defined. Then, primers are positioned to obtain all target regions

as the smallest possible number of fragments that can still be amplified through Iong-

PCR, i.e. up to at least 20 kb (TaKaRa LA PCR Kit Product Insert). Some general

guidelines in choosing primer sequences for Iong-PCR have been described (Foord and

Rose, 1994, Long-distance PCR, PCR Methods and Applications 3, S149-S161 ), but

empirical determination of optimal primers remains necessary.

Table 1. Design of a TDGS test. 1. Retrieve sequence from database.

2. Position primers for loπg-PCR to cover all desired regions (e.g., coding sequences, splice sites, regulatory regions, mutation hotspots) by the smallest possible number of amplicons.

Position primers for short-PCR according to the following criteria: a. the desired target sequences should be covered by amplicons of between 100 and 600 bp b. amplicons should have optimal melting behaviour, i.e., consist of one lowest- meitiπg domain in addition to the GC-clamp attached to one of the primers. c. optimal amplicoπ distribution over the 2-D gel d. similar reaction kinetics

Set up PCR conditions separately for each primer set with the Iong-PCR products as template.

5. Develop multiplex co-amplification conditions by grouping primer sets and adjusting reaction components. As listed in Table 1 , item 3, then, using the loπg-PCR fragments as template,

primers for short-PCR are selected to yield fragments of between 100 and 600 bp. The

main selection criterion here is πecessariiy the melting behaviour of the fragments. In the

ideal situation, each ampiicoπ should comprise oπiy one meitiπg domain, which should

be lower (less stable) than the GC-clamp attached to it. Attachment of a 30-40 bp GC-

clamp is accomplished by making it part of one of the primers (Sheffield et al., 1989, The

sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions, Genomics 16, 325-332). Optimal meitiπg behaviour is determined of each candidate target sequence by using a computer program (e.g., MELT87; Lerman and Silverstein, 1987, Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis, Meth. Enzvmol. 155, 482-501). An example of an ampiicoπ with optimized meitiπg behaviour through GC-ciamping is shown in Rg. 2, in connection with exon 12 for RB.

In general, a collection of primers is selected that allow an optimal distribution, in both size and DGGE dimension, over the 2-D gel. Due to the high resolution of 2-D gels

(5-10-bp size differences are easily resolved) this is generally not too difficult. Indeed, with

50 fragments or less, spot distribution is hardly an issue and primers can simply be selected according to their melting behaviour.

Rg. 3 shows the collection of amplicons selected for the RB gene, together with the Iong-PCR fragments that served as templates. Together the short-PCR fragments represent more than 90% of the RB coding region.

Rgs. 4 and 5 show the theoretical and the empirical spot distribution for the 24 exons of the RB gene covered by the ampiicoπs shown in Rg. 3. Although there are

differences, most notably the spot representing exon 11 , our conclusion is that overall the

meitiπg program accurately predicts spot positions.

It is important to realize that without the first loπg-PCR step, the optimal meitiπg

criterium is usually in conflict with other primer design criteria applied to PCR with total

genomic DNA as template. Indeed, for the RB gene it was found to be impossible to

select conditions suitable for both optimal separation in DGGE and optimai priming in

PCR. The pre-purificatiαn step represented by the Iong-PCR is apparently a coπditio sine qua non for the design of an optimal set of PCR primers in TDGS. When the test format is established, the two-step PCR amplifications are carried out in a multiplex format. It is the possibility to design and perform multiplex PCR reactions that represents the core of the present invention. The necessity of the first loπg-PCR step for a successful multiplex PCR is demonstrated by the results shown in Rg. 6. In Rg. 6, the 6 lanes on the left contain PCR products obtained after performing a multiplex PCR of exons 18-23 (6 fragments) of the RB gene, using different amounts of total genomic DNA as template. Clearly, virtually no products ofthe desired lengths are obtained. The latter is in contrast to lanes 7-12, in which the products were applied of the same multiplex PCR reaction, but this time with the Iong-PCR product as template. The loπg-PCR was performed at different cycles and it is dear that only 5-10 cycies are needed to generate enough template for a successful multiplex PCR.

Lanes 13 to 19 contain the multiplex short-PCR products obtained with the loπg- PCR products as template, at different amounts of starting material, i.e., different amounts of total genomic DNA used in the loπg-PCR reaction. Interestingly, 5 ng total genomic

DNA is sufficient to obtain ail the products. Since clinical material is sometimes not

available in plentiful amounts (e.g., breast cancer needle biopsies) this is an important

result, indicating that a successful test can be performed with very small amounts of DNA.

Rnally, lanes 20-24 of Rg. 6 contain the products of the 5 multiplex PCRs

corresponding to the 6 loπg-PCR sets (Iong-PCR groups 1 and 6 were combined; see also Rg. 3 and later discussed Table 2). Further adjustments of PCR conditions and/or primers should make it possible to obtain an even smaller number of multiplex sets for this gene. Indeed, there is no reason why the entire RB gene coding region couldn't be amplified in only one single PCR reaction. After the second PCR, the fragments are allowed to undergo one complete round of deπaturatioπ/reπaturatioπ to facilitate the formation of heterodupiexes. There are presented in Table 2, a listing of the primer pairs for TDGS for the case of RB; the exon numbers being listed in the left most table, with long PCR primer codes for six exon groups (0 through 24-27) and short PCR primers for the individual 27 exons.

Subsequent to the PCR, the mixture of fragments is subjected to 2-D electrophoresis in a denaturing gradient gel (Rg. 1). The availability of an automated instrument greatly simplifies this process. The instrument used here allows 10 gels at a time to run without manual interference, i.e., cutting out lanes and loading these onto a second gel. All experiments involving optimization of the experimental conditions were carried out using manual instruments. After 2-D electrophoresis the gels are released from between the glass plates and stained with ethidium bromide or any other stain. Table 2. Primer pairs for TDGS of RB

RB: Long ;<-PC exons primers 5'— 3' size

0-2 TGTCAGGCCTGCCTGACAGACTTCTATTCAGCA 4.5 kb ATGTΓAGCAGAGGTAAATTTCCTCTGGGTAATGG

3-6 GCAGTCATTTCCCAACACCTCCCCTCTGT 9 kb

AAGCCAAGCAGAGAATGAGGGAGGAGTACATTAC

7-11 TC^G ^GTTTCTCCCTCCAAGTCAGAGAGGC 10 kb GAGACCAGAAGGAGCAAGATCAGGTAGTAG

12-17 ACCATTCCCCCTACTCTCCATGGTCCATG 12.4 b CTCACAGGAAAAATACACAGTATCCTGTTTGTGTGGC

18-23 CCAGCCTTGCATTCTGGGGATGAAGC 14 kb

AGTCGTAAATAGAITI 1 1 1 CACCCCGCCCC 24-27 GCCTΓTGCCCTCCCTAAATATGGGCAATGG 7.3 kb CTGGGTTATCAGGACTCCCACTCTAGGGCC

RB: Short- PCR exon primers 5'— 3'

[GCI] TTGATTTATAAGTATATGCCA CAAAACGTTTTAAGAAAATCC

[GCI] CCAGTGTGTGAATTATTTAA CCTTTTATGGCAGAGGCTTATA

4 [GCI] GAATTGAAATATCTATGATT

ATCAGAGTGTAACCCTAATA 5 [GCI] TACTATGACTTCTAAATTACG

GTGAAAAATAACATTCTGTG

6 TGGAAAAC1 1C11 lCAGTG

[GCI] GAATTTAGTCCAAAGGAATGC

[GCI] CCTGCGATTTTCTCTCATAC GCAACTGCTGAATGAGAAAG

GTTCTΓATCTAATΓTACCACT [GCI] TTTTAAAGAAATCATGAAGTT

9 [GCi] AGTCAAGAGATΓAGATTTTG

ATCCTCCCTCCACAGTC 10 [GCI] GACATGTAAAGGATAATTGT

GCAAATCAATCAAATATACC 17/1

11 AGTATGTGAATGACTTCACT 174bp 21 B

[GCI] TATAATATAATTAAAAGTAGG

CTCCCTTCATTGCTTAACAC 211bo 24 [GCI] TTTCTTTGCCAAGATATTAC

13 [GCI] GATTACACAGTATCCTCGAC 224bp 34 GCAGTACCACGAATTACAATG 14 [GCI] GTGATTTTCTAAAATAGCAGG 179bp 35 ACCGCGCCCGGCTGAAAT

17 [GCI] TTCTTTGTCTGATAATAAC 380bp 26 C CTCACTAACAATAATTTGTT

18 [GCI] GACTTTTAAATTGCCACTGT 393bp 33 ATTCCCTACAG l TC lTAT

19 [GCI] CAACTTGAAATGAAGAC 248bp 34 CGTCCCGCTGCTCTTGAAAATAATCATC

20 [GCI] AAAATGACTAA1 1T1 1 1 lATTCCC 227bp 44 AGGAGAGAAGGTGAAGTGC 21 [GC2] CATTCTGACTACTTTTACATC 201bp 28 CGGGCTTACTATGGAAAATTAC

22 [GC3] CTTTTTACTGTTCTTCC 194bp 33 CCAATCAAAGGATACTTTTG

23 [GCI] TCTAATGTAATGGGTCCACC 2Slbp 38 CCCTACTTCCCTAAAGAGAAAAC

24 CGGAATGATGTATTTATGCTCA 195bp 22 [GCI] TTCTTTTATACTTACAATGC

25 [GCI] ATGATTTAAAGTAAAGAATTCT 245bp 38 CATCTCAGCTACTGGAAAAC

26 [GCI] TCCATTTATAAATACACATG lύlbp 32 ATTTCGT7TACACAAGG7G

27 [GCI] TACCCAGTACCATCAATGC 191bp 43 TCCAGAGGTGTACACAGTG

GC- iamps:

GCI: CGCCCGCCGCGCCCCGCGCCCGTCCCGCCC (30mer) GC2: CGCCCCGCGCCGCCGCCCCGCCCCCGCCCGTCCCGCCC (38mer) GC3: CGCCCCGCCGCGCCCCGCGCGCCCGGTCCCCGCGC (35mer;

The resulting patterns were documented and evaluated (by eye and image analysis ) for the occurrence of mutations. Under the conditions applied ie, GC-clamping and heteroduplexing, heterozygous mutations result in 4 spots: the 2 homoduplex variants 18 and the 2 heteroduplex variants (illustrated in Rg.7). The latter are not always separated.

Since mutations may also occur in a homozygous state it can be necessary to mix each

sample before PCR with a control sampie to make sure that heteroduplex molecules will

be present.

The following provides detaiis of the manner in which the embodiments of the

present invention may be made and used in order to achieve the accurate and efficient

preparation and examination of gene fragments in DNA diagnostic testing. This

description, which is focused on an illustrative or model gene, i.e., the tumor suppressor gene RB previously discussed, is not to be construed as specifically limiting the invention. The same procedure may be used on other genes and/or to combine even more PCR fragments in the same tube, within the purview of one skilled in the art, and are to be considered to fall within the scope of this invention.

A. Design of the RB Two-Step PCR TDGS Test

Sequence Retrieval. The sequence of the RB gene is retrieved from a database, i.e., Genbank. The target regions, i.e., exons, splice sites, regulatory regions are defined.

Primer Selection for multiplex Long-PCR. Primer pairs for loπg-PCR are positioned in such a way as to cover all target regions by the smallest possible number of fragments that can still be amplified through loπg-PCR. Long-PCR primers are also selected for highest specificity, optimal annealing temperature and minimal self-complementation for 19 multiplex loπg-PCR, e.g., by using primer design software. The design of the Iong-PCR

multiplex is relatively easy in view of the ample positioning space of the primers.

Primer Selection for Multiplex Short-PCR. Primer pairs for short-PCR are selected

on the basis of the following criteria:

a. the desired target sequences should be covered by amplicons of between 100 and 600 bp

b. amplicons should have optimal meitiπg behaviour, i.e., consist of one lowest- melting domain in addition to the GC-ciamp attached to one of the primers.

c. optimal ampiicoπ distribution over the 2-D gel

d. similar reaction kinetics

Criterion b, above, is frequently in conflict with standard primer design criteria, if applied on total genomic DNA. Indeed, the present invention proved to be both necessary and sufficient for the design and performance of TDGS as a rapid, accurate and practical tool for mutation detection in the RB gene.

Multiplex groups are selected empirically on the basis of the behaviour of the primers in various multiplex reactions. For RB multiplex groups were made according to 20 the loπg-PCR. That is, all short PCRs with one loπg-PCR fragment as template were

amplified together as one multiplex group. Two loπg-PCR groups were actually combined

as one multiplex group. All short-PCR fragment may also be amplified together. As before

explained, Table 2 lists the primer pairs used for the long and short PCRs, fragment

sizes, annealing temperatures, meitiπg temperatures and the five different multiplex

groups.

B. PCR Reactions and Heteroduplexing

Primers (deprotected and desalted) can be obtained from various sources. Our primers were obtained from Gibco BRL For long-term storage, primers should be kept for example in a stock solution of 100 μM in uitrapure water, at -20 °C. For short-term use, we kept them at -20 °C as a solution of 12.5 μM in uitrapure water.

We carried out our PCR reactions in thermowell tubes (Costar, Cambridge, MA) in a GeπeE thermocycier (Techπe, Cambridge, UK) fitted with a Heated Lid, removing the need for an oil overlay on the samples. Multiplex long PCR reactions (6 fragments) were carried out in a 100 μl volume with 5-500 ng genomic DNA as template and 0.2 μM of each primer, using the LA PCR kit (TaKaRa). PCR reactions are performed according to the manufacturer's instructions. The conditions were as follows. Rrst, one cycle of 94 °C, 1 min, followed by 30 cycles of 98 °C, 20 sec / 68 °C, 12 min with 10 s increment per cycle, and finally one cycle of 72 °C, 12 min. The PCR products are stored at -20 °C for further use. 21 Short PCR reactions are carried out, using the same GeneE thermocycier, in a 50

μl volume with 2 μl loπg-PCR product, 0.2-0.5 μM of each primer, 0.25 mM dNTPs, 2.5-

4.5 mM MgCI₂, 3 units of Taq polymerase (Gibco BRL or Promega). The PCR conditions

are as follows. One cycle of 94 °C, 2 min, then 30 cycles of 94 °C, 40 sec / 41 °C, 40 sec

/ 69 °C, 2 min (with a 2-sec increment increase per cycle) and finally one cycie of 72 °C,

10 min.

After the short PCR, fragments are heteroduplexed by one complete round of

deπaturatioπ reπaturation. That is, 98°C, 10 min / 55 °C, 30 min / 41 °C, 30 min.

After PCR and heteroduplexing the contents of the tubes are mixed and 1/10 volume of loading buffer is added. Based on ethidium bromide staining, there is usually enough sample for several runs. When the total volume is too large for the slot capacity, the sample (prior to adding loading buffer) has to be ethaπol-precipitated and re-dissolved in a smaller volume.

C. Two-Dimensioπai Electrophoresis.

Instruments for both manual and automatic 2-D electrophoresis were from the before mentioned Ingeny B.V. (Leiden, The Netherlands). For manual electrophoresis, the mixtures of DNA fragments were first subjected to size separation using a 0.75 mm thick 9% PAA gel at 45 °C for 5-6 h. The separation pattern was visualized by ethidium bromide staining for 10 min and UV transiilumiπatioπ of the gel, which lies on a glass plate to protect the DNA fragments from damage by the UV light The 100 to 600 bp 22 region in the middle part of the lane (so not including the edges) was quickly cut out and

applied to a 1-mm thick 9% PAA gel containing a 0-60% (RB) or 30-90% (p53)

urea/formamide (UF) gradient Gradients were poured using a simple gradient former

(Gibco BRL). Electrophoresis was for 7.5 - 11 h at 60 °C and 200 V. After electrophoresis

the gels were stained with 0.5 μg/ml ethidium bromide for 15-20 min and destained in

water for another 15 min. The patterns were documented under UV illumination using a

Polaroid camera.

For automatic 2-D electrophoresis, gels were poured, ten at a time, in the gel-

casting device that comes with the automated 2-D electrophoresis instrument according to the manufacturer's instructions (Ingeny B.V., Leiden, The Netherlands). After polymerization the gels (between glass plates) are removed from the gel-casting box and cleaned with a wet tissue. They are then placed in the instrument according to the manufacturer's instructions, that is, in two gel-holding cassettes with siiicone-side seaiiπgs. The instrument containing buffer heated to 45°C is put in the 1-D mode with the power switched off. After adding loading buffer, samples (up to 40 μl) are loaded in the V-shaped wells of the gels in the automated 2-D electrophoresis instrument. Gels of 9% acrylamide, 0.25 % TAE were used with a gradient of 0-60% urea/formamide. The first dimension is run at 180 V for 4 h at 45°C. The second dimension was run at 200 V for 7.5 - 11 h at 60 °C. After electrophoresis the gels were stained with ethidium bromide and the patterns documented under UV illumination as described for the manual instruments. 23 In summary, while the present invention is described in connection with two-step

(long and short) multiplex polymerase chain reaction amplifications followed by two-

dimensional electrophoretic separation, such is also useful with one-dimensional

electrophoresis or with other methods for mutation detection that require PCR-amplified

target sequences.

Further modifications wiil also occur to those skilled in this art and such are considered to fail within the spirit and scope of the invention as defined in the appended

claims.

Claims (10)

24CLAIMS

1. A method of analyzing predetermined gene exons derived from DNA, that

comprises, adding primer pairs to successive groups of the gene exons followed by effecting polymerase chain reaction amplifications thereof in a common tube, as a first

step and relatively long multiplex polymerase chain reaction; adding further primer pairs

to each of the gene exons and effecting polymerase chain reaction amplifications thereof in the common tube as a second step and short multiplex polymerase chain reaction; and electrophoretically separating the gene fragments.

2. A method as claimed in claim 1 and in which the gene fragments are electrophoretically separated on the basis of size along one dimension.

3. A method as claimed in claim 2 and in which the gene fragments are further subjected to further electrophoretic base pair sequence separation along an orthogonal dimension and along a temperature or chemical denaturing gradient to distribute the gene fragments at particular sequence locations along the orthogonal dimension; and comparing such locations with those of normal gene fragments to detect genetic mutations.

4. A method as claimed in claim 3 and in which the primers are oligonucleotide primers labelled with fluorescent dyes.

5. A method as claimed in claim 3 and in which the nucleotide building blocks used in the polymerase chain reaction are fluorescently labelled.

6. A method as claimed in claim 3 and in which the electrophoretic separation is effected in a denaturing gradient gel. 25

7. A method as claimed in claim 6 and in which a gradient of urea and formamid

is applied in a gel to separate the fragments.

8. A method as claimed in claim 6 and in which a temperature gradient is applie

in a gel to separate the fragments.

9. A method as claimed in claim 3 and in which, for the retinobiastoma gene, th loπg-PCR gene exon groups are exons 1-2, 3-6, 7-11, 12-17, 18-23 and 24-27.

10. A method of analyzing predetermined gene exons derived from DNA, th comprises, adding primer pairs to successive groups of the gene exons followed b effecting polymerase chain reaction amplifications thereof in a common tube, as a fir step and relatively long multiplex polymerase chain reaction; and adding further prim pairs to each of the gene exons and effecting polymerase chain reaction amplification thereof in the common tube as a second step and short multiplex polymerase chai reaction.