WO1997031012A1

WO1997031012A1 - Gene specific universal mammalian sequence-tagged sites

Info

Publication number: WO1997031012A1
Application number: PCT/US1997/002403
Authority: WO
Inventors: George J. Brewer; Patrick J. Venta; Vilma Yuzbasiyan-Gurkan
Original assignee: The Regents Of The University Of Michigan; Board Of Trustees Operating Michigan State University
Priority date: 1996-02-22
Filing date: 1997-02-18
Publication date: 1997-08-28
Also published as: AU1959897A

Abstract

Primer sets which amplify conserved regions of specific genes across mammalian species are provided. The methods used to design the primer sets as well as methods of making and using the primer sets are also provided.

Description

GENE SPECIFIC UNIVERSAL MAMMALIAN

SEQUENCE-TAGGED SITES

FIELD OF THE INVENTION

The present invention relates generally to genetic markers and methods of making and using such markers, and more particularly, to primer sets referred to as universal mammalian sequence-tagged site primers, which may be used to amplify conserved regions of specific genes across mammalian species.

BACKGROUND OF THE INVENTION

Over the last several years significant effort has been made to develop genome maps of many species. The subjects of the majority of these projects have been mammals, including human, mouse, rat, ox, sheep, pig, horse, cat, and dog (e.g. , Buchanan F.C. et al. , Genomics 22: 397-403 (1994); Dietrich, W. et al. , Genetics 131 : 423-447 (1992); Ellegren, H. et al. , Anim. Genet. 23: 133-142 (1992); O'Bnen, S. J. , Trends Genet. 2: 137-142 (1986); Serikawa, T. et al. , Genetics 131: 701-721 (1992); Weissbach, J. et al. , Nature 359 794-801 (1992), Wintero, A.K et al. , Genomics 12: 281-288 (1991); and Barendse, W. et al. , Nat. Genet. 6:227-235 (1994)). For non-human mammals, genome maps will lead to important applications including optimized breeding strategies, wherein desirable characteristics are selected and unwanted genes are removed, particularly those genes that lead to genetic disease . Comparisons made between genome maps will also provide a wealth of scientific information (O'Brien, S. J. et al., Nat. Genet. 3: 103-1 12 (1993)) .

The traditional method for developing gene-specific markers, Southern blotting and cross-species hybridization, is time consuming, labor intensive and limited in flexibility. However despite its many limitations, this method remains the primary method for developing gene-specific markers in most mammalian genome projects. There thus remains a need for a more efficient method. A more efficient method is particularly important for non-human mammalian genome projects wherein scientific resources are often limited .

One potential method is the development of polymerase chain reaction (PCR) primers to conserved regions of specific genes that can be used across species. Although this method has been successfully used for the study of a number of individual genes, it has not been applied on a genome-wide basis for the purpose of map development.

It would thus be desirable to provide genetic markers which may be used to detect specific mammalian genes. It would further be desirable to provide genetic markers which may be used to develop mammalian genome maps. It would further be desirable to provide genetic markers which may be used across mammalian species. It would also be desirable to provide a method for designing and producing genetic markers based on conserved regions of genes.

SUMMARY OF THE INVENTION

Primer sets which may be used to amplify conserved regions of specific genes, including eleven preferred primer sets that have been shown to amplify specific canine genes, are provided. The methods used to design the primer sets as well as methods of making and using the primer sets for example with a polymerase chain reaction (PCR), are also provided. Such genetic markers based on PCR primers are often called sequence-tagged sites (STSs) or sequence-tagged site primers (Olsen, M. et al., Science 245:1434-1435 (1989)). Because the primer sets of the present invention may be used to locate genes across mammalian species, such primer sets are referred to herein as universal mammalian sequence- tagged site (UM-STS) primers.

Additional objects, advantages, and features of the present invention will become apparent from the following description and claims taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and claims and by referencing the following drawings in which:

Figure 1 is a photograph of a gel showing the amplification of several canine gene segments using the UM-STS primers of the present invention;

Figure 2 shows the lineup of several canine gene sequences with homologous non-canine mammalian genes;

Figure 3 is a photograph of a gel showing the amplification of a portion of the FES proto-oncogene from several mammalian DNAs using the UM-STS primers of the present invention; and

Figure 4 shows sequence comparisons of a portion of the FES proto-oncogen from several mammalian DNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The universal mammalian sequence-tagged site primer sets of the present invention are set forth in Tables 1 and 1A. The methods used to design the primer sets as well as methods of making and using the primer sets are also provided. The primer sets of the present invention referred to herein as UM-STS primers, may be used to amplify mammalian genome regions of interest, isolate clones from mammalian genomic and cDNA libraries and perform cross-species genome comparisons. The UM-STS primers of the present invention will also be useful for developing additional genetic markers within various genomes. For example, a microsatellite repeat has been found within the retinoblastoma (RB1) loci disclosed herein. Single site variability may also be found directly in at least some of the amplified products by using one of a number of techniques developed for scanning for variability, such as the single-strand conformation polymorphism technique. For example, this method has been used to find two polymorphic sites in a study of the canine ALAS2 gene in a PCR product of a size similar to those set forth herein (Boyer, G. et al., Anim. Genet. 26:206-207 (1995)). If the frequency of single site polymorphic variability for other mammals is as high as that estimated for humans (roughly one in 200 to 400 nucleotides), then a significant portion of UM- STSs will have these sites. Each species will be screened individually for genetic variability using UM-STS primers.

An example of the usefulness of cross-species genome comparisons is given by the case of Waardenburg syndrome. Direction to the location of one of the human Waardenburg syndrome genes, well known for causing a syndromic hearing loss, was first found from comparative mapping with the mouse (Asher, J.H.J. et al., J. Med. Genet. 27:618-626 (1991)). The map locations in the mouse suggested possible locations of the human disease gene, one of which eventually was proven correct (Morrell, R. et al., Hum. Mol. Genet. 156:53-59 (1993)). Because the identity of the gene in the mouse was not known at the time, this approach might more properly be called a 'candidate linkage' approach. UM-STSs will thus be useful not only for rapidly producing genetic maps but the candidate linkage approach may also be applied to more species.

Very little is known about the location of genes within the canine genome. The development of UM-STSs will help to rapidly identify the location of linkage groups on specific canine chromosomes. The assignment of conserved syntenies will allow candidate linkages to be tested in the canine genome. The assignment of the proposed anchor loci (O'Brien, S.J. et al., Nat. Genet. 3:103-112 (1993)) as defined by UM-STSs to specific chromosomes may be accomplished by the somatic cell hybrid, flow sorted chromosome, and fluorescent in situ hybridization (FISH) methodologies. Other methods known to those skilled in the art, such as assignment by use of linkage to previously mapped loci, are also possible.

SPECIFIC EXAMPLE 1

The following example further describes the UM-STS primers of present invention as well as methods of designing, making and using such primers.

Materials and Methods

DNA Isolation - DNA from dog, human, pigtail macaque, horse, pig, rat and mouse were isolated from various tissues by standard phenol-chloroform extraction methods (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual. (2nd ed.) Cold Springs Harbor: Cold Springs Harbor Laboratory Press (1989)). Goat DNA was supplied by Dr. Karen Friderici, Michigan State University. DNA was purified by standard methods from a canine liver cDNA library (Clontech) and from a canine genomic DNA library (Clontech) after growing 1 x 10⁶ phage in E. coli strain LE392 (Murray, N.E. et al., Mol. Gen. GeneM56:53-59 (1977)) in liquid culture (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual. (2nd ed.) Cold Springs Harbor: Cold Springs Harbor Laboratory Press (1989)).

Design of PCR Primers - Primers were designed to genes where the intronexon structure was known in at least one species and where the nucleotide sequence was known in at least two species (the index species) that were not closely related. Tandemly duplicated genes known to have undergone gene conversion in any species were avoided. Primers were generally designed so that the amplified product contained an intron. The human gene nomenclature system (ISGN, Cytogenet. Cell Genet. 46:11-28 (1987)) was followed for naming the canine genes. An example of the loci used and their protein products are: CFTR, cystic fibrosis transmembrane regulator; COL10A1, type X collagen, alpha 1 chain; CSF1R, colony stimulating factor 1 receptor; CYP1A1, cytochrome P-450 1 , alpha 1 ; DCN1, decorin; FES, c-fes (feline sarcoma) proto-oncogene; GHR, growth hormone receptor; GLB1, beta galactosidase; PKLR, pyruvate kinase - liver, RBC form; PVALB, parvalbumin; and RB1, retinoblastoma protein. The Genbank Accession numbers or reference for the sequence of the two index species for each locus are as follows: CFTR, M55129, M60493; COL10A1, X65120, X65121 ; CSF1R, X14720, K01643; CYP1A1, (Uchida, T. et al., Mol. Pharmacol. 38:644-351 (1990)), X04300; DCN1, L01125, Z12298; FES, X06292, J02088; GHR, Z11802, J04811 ; GLB1, S59584, M57734; PVALB, X63578, M15452; PKLR, S59798, M17088; and RB1, L11910, M26391. Primers were designed to highly conserved nucleotide sequences contained within coding regions. Additional considerations taken into account were: degeneracy of underlying codons (Li, W.-H. et al., Fundamentals of Molecular Evolution. Sunderland, MA: Sinauer Associates, Inc. (1987)), overall amino acid mutability of the primer region (Collins, D.W. et al., Genomics 20:386-396 (1994)), placement of the 3' end of the primer with respect to amino acid mutability, as well as standard design practices for primers known in the art such as avoidance of primer-dimers. Conservation of amino acids within multigene families was also taken into account, when possible. Where unavoidable mismatches occurred between the two index species, the primer sequence was designed to match one of the two which was then designated the primary index species. GC-rich genes were avoided due to amplification difficulties, even with exactly matching primers. Primers were twenty bp in length on average. Each primer in a pair was adjusted to be of approximately the same annealing temperature (Breslauer, K. et al., PNAS (USA) 83:3746-3750 (1986)). All sets of primer pairs were designed to have approximately the same annealing temperature in anticipation of performing multiplex amplifications. It was not always possible to follow every rule for every gene, given the actual circumstances; however, the majority of the rules were generally applicable.

PCR Amplifications - Correct design and syntheses of the primers were examined by amplifying the DNA from the primary index species. Standard buffer, nucleotide, and primer concentrations were 50 mM Tris-HCI (pH 8.3 at room temperature), 50 mM KCI, 1.5 mM MgCI₂, 200 μM dNTPs, 0.1 μg of each primer, and 0.5 - 1.0 μg of target DNA in a 25 μl reaction. Reactions were routinely boiled for three min prior to the addition of 2.0 U of Taq DNA polymerase. Optimal cycling conditions for the amplification of canine genomic DNA were usually found by testing one of several sets of conditions in general use in the lab (see Table 2). Occasionally it was necessary to use "hot-start" conditions (Bassam, B.J. et al., Biotechniques 14:30-34 (1993)) in order to get stronger, cleaner amplifications. The presence of an amplification product was determined by electrophoresis of a portion of the reaction on a 1% agarose 1X TBE gel (90 mM Tris, pH 8.3, 90 mM sodium borate, 2.5 mM EDTA) followed by staining with ethidium bromide.

DNA Sequence Analysis - The identity of each amplified canine gene was confirmed by 'single pass' direct sequencing of PCR products using Sequence or Taq DNA polymerase (United States Biochemical Corp., Cleveland). The PCR products were gel purified with Qiaex (Qiagen Corp., Chatsworth, CA) or by elution from polyacrylamide gel slices (Bergenhem, N.C.H. et al., Biochem. Genet. 30:279- 287 (1990)) prior to their use in the sequencing reactions. The canine sequences were aligned visually with the sequences of the other species used to design the PCR primers in order to verify the degree of sequence identity.

Results

The primer sets for the various UM-STSs reported here are given in Tables 1 , 1A and 2 and efficient amplification conditions for the eleven preferred UM-STSs are given in Table 3. It will be appreciated by those skilled in the art that the conditions may be optimized further (e.g., reduction in the time in each cycle). However, the conditions set forth herein were found to work effectively while minimizing the number of conditions that had to be examined.

A representative gel showing amplification of the canine target DNA along with the human target DNA is shown in Figure 1. In Figure 1 the various lanes were amplified with the following gene-specific primer sets of the present invention: lanes 1-4, GHR, lanes 5-8, COL10A1, and lanes 9-12, DCN1. Lane 13 contained a mixture of DNA size markers; lambda bacteriophage DNA cut with the restriction endonuclease BstE II and the plasmid pSK- (Stratagene) cut with Msp I. Lanes 1 , 5, and 9 contain PCR products amplified from human genomic DNA. Lanes 2, 6, and 10 contain PCR products amplified from canine genomic DNA. Lanes 3, 7, and 11 contain PCR products amplified from DNA purified from a canine genomic library contained in a lambda phage vector. Lanes 4, 8, and 12 contain PCR products amplified from a canine liver cDNA library. The human target serves as a positive control for the amplification system because these primers were designed to exactly match the human sequence. The ability to quickly screen genomic and cDNA libraries for the presence of sequences is also demonstrated in Figure 1. The genomic clones for GHR, COL10A1, and DCN1 (a very faint signal, stronger on other gels [data not shown]) are present in this particular canine genomic library. The presence of a decorin cDNA clone (encoded by the DCN1 locus) in the canine liver cDNA library is shown by the presence of the 122 bp band; cDNA clones for GHR and COL10A1 are not present. The DCN1 PCR product from the cDNA library was sequenced and its identity confirmed (see Figure 2). The human and canine genomic bands have different sizes for GHR and DCN1 because of the intron size differences. The size for the COL10A1 PCR product is the same between the species because this the only perferred UM-STS set forth herein in which an intron was not spanned. Although the PCR product bands in Figure 1 are unique, some UM-STS-species combinations contained one to several non-specific amplification products. However, it is almost always possible to deduce the correct band based upon staining intensity and the similarity in size compared to the band of the primary index species.

The amplified products for all of the canine loci (see Table 2) were sequenced to confirm their identity and the results are shown in Figure 2. The locations of PCR primers are underlined, although not all PCR primer sites are shown. Some of the lineups show intron sequence whereas others simply identify the location of the introns. The degree of identity between the canine and index species sequences for each locus is within the range generally accepted (approximately 70 to 100%) as indicating homology for mammalian species (Li, W.- H. et al., Fundamentals of Molecular Evolution. Sunderiand, MA: Sinauer Associates, Inc. (1987)). These results support the hypotheses that the canine PCR products are homologous to the respective index species' genes. The canine COL10A1 sequence matched the human and mouse sequences to a similar extent (data not shown). The sequences for PKLR and CYP1A1 exactly matched previously published canine coding sequences (Whitney, K.M. et al., Exp. Hematol. 22:866-874 (1994); and Uchida, T. et al., Mol. Pharmacol. 38:644-351 (1990)); the sequence for canine FES is shown in Figure 4. Although the majority of the canine sequence for PVALB is from an intron, it is believed that the degree of sequence identity from this region is sufficient evidence to confirm that the PCR product is from the correct canine locus. As expected, the canine sequences tend to show greater identity with the human sequences than with the rodent sequences because of the faster evolutionary rate of the rodent genome (Gu, X. et al., Mol. Phylogen. Evol. 1:211-214 (1993)). As set forth above, a microsatellite repeat was found within the amplified product itself for RB1. Preliminary results show that the RB1 repeat, (GA)_12(avg), has moderate genetic variability within several canine breeds.

The 'universal' utility of these primers was studied on the DNAs from mammals representing several different orders using the primer sets under the reaction conditions that were found to amplify the canine sequences. These reactions are referred to herein as 'Zoo PCRs.' Figure 3 shows a representative experiment. The FES proto-oncogene amplified from all of the mammalian species were examined. These DNAs were purified and sequenced directly without subcloning (see Methods and Materials above). In Figure 3, target DNAs for each lane are: 1 , human; 2, pigtailed macaque; 3, dog; 4, goat; 5, pig; 6, horse; 7, mouse; and 8, rat. The mouse DNA was degraded; strong amplification was obtained with another lot (sequence shown in Figure 4). The DNA marker lane (M) contains a 100 bp ladder. The sequences are tabulated in Figure 4. Sequences are from exon 15 and intron 15. Notations for the sequence lineups in Figure 4 are HUM, human; MAC, macaque; CAT, domestic cat; FES, feline sarcoma virus; DOG, dog; COW, ox; GOA, goat; HOR, horse; PIG, pig; RAT, rat; MOU, mouse. The upper two lines for each block of text represent amino acid sequences and the lower lines represent nucleotide sequences. Dots indicate nucleotides in the various species that are identical with that of the human sequence. The human and cat sequences match the published sequences (Alcalay, M. et al., Oncogene 5:267-275 (1990); and Roebroek, A.J.M. et al., J. Virol. 61 :2009-2016 (1987)). The feline sarcoma virus sequence was not determined but is included for comparative proposes. Only a single amino acid interchange was found among these sequences; isoleucine (I) for macaque, cat, and feline sarcoma virus and leucine (L) in all others. Sequence alignments for the intron were done visually.

The degree of sequence identity makes it highly likely that the canine PCR products are homologous with the corresponding index species' genes. The pattern of nucleotide interchange is also what would be expected for homologous genes; members of the same mammalian order share more sequence similarity with one another than with those of other orders.

The data for the Zoo PCRs for the preferred UM-STS primer sets are given in Table 4. Greater than eighty-four percent of the targets, excluding the index and canine species, amplified under the single condition used to amplify the canine sequence. These species represent five different mammalian orders; primates (human and macaque), carnivores (dog), arteriodactyls (goat and pig), perissodactyls (horse), and rodents (mouse and rat). Limited experiments on other members of these orders (e.g., cat and ox) produced similar results (data not shown). Lack of amplification for DCN1 for one of the arteriodactyls (goat) would be predicted because there are four mismatches between the UM-STS primers and the sequence of the closely related bovine DCN1 (Day, A.A. et al., Biochem. J. 248:801-805 (1987)).

The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion and from the accompanying claims and drawings, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention.

All publications referred to herein are expressly incorporated by reference.

Claims

WE CLAIM:

1. A primer comprising a polynucleotide, wherein the polynucleotide has a sequence selected from the group consisting of the sequences set forth in Table 1.

2. A primer comprising a polynucleotide, wherein the polynucleotide has a sequence selected from the group consisting of the sequences set forth in Table 1A.

3. A method for amplifying DNA, comprising the step of performing PCR with the DNA and a primer set selected from the group consisting of the primer sets of Table 1.

4. A method for amplifying DNA, comprising the step of performing PCR with the DNA and a primer set selected from the group consisting of the primer sets of Table 1A.