Bidirectional PCR amplification of specific alleles

Details Bidirectional PCR amplification of specific alleles Bidirectional PCR amplification of specific alleles

1998-09-10

2001-03-27

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

Reexamination Certificate

active

06207425

ABSTRACT:

BACKGROUND OF THE INVENTION
This is an invention the field of amplification of DNA by polymerase chain reaction (PCR).
PCR is a method that typically utilizes two oligonucleotide primers to amplify a DNA segment >1 million-fold. The two primers anneal to opposing strands of DNA at positions which span a target sequence of interest. A DNA polymerase is used for sequential rounds of template dependent synthesis of the DNA sequence. The PCR method is more fully described in U.S. Pat. No. 4,683,202, issued Jul. 28, 1987, the disclosure of which is incorporated herein.
PCR can be adapted for the rapid detection of single-base changes in genomic DNA by using specifically designed oligonucleotides in a method called PCR amplification of specific alleles (PASA) (Sommer et al. 1989; Sarkar et al. 1990; Wallace et al. U.S. Pat. No. 5,639,611, issued Jun. 17, 1997). This rapid method is also known as allele-specific amplification (ASA), allele-specific PCR, and amplification refractory mutation system (ARMS) (Newton et al. 1989; Nichols et al. 1989; Wu et al. 1989). For this technique an oligonucleotide primer is designed to match one allele perfectly but mismatch the other allele at or near the 3′ end, thereby preferentially amplifying one allele over the other. PASA assays can be developed for assaying virtually all alleles (Sommer et al. 1992). However, each PASA reaction provides information on the presence or absence of only one allele. Two PASA reactions must be performed to determine the zygosity of any sequence change.
To detect zygosity in one PCR reaction, PCR amplification of multiple specific alleles (PAMSA) utilizes three primers in one reaction to generate two allele-specific segments that differ sufficiently in size to be distinguished by agarose gel electrophoresis (Dutton and Sommer 1991). However, problems arise from PAMSA because of differences in length, and hence, amplification efficiency of the allele-specific primers. A similar approach-termed competitive oligonucleotide primary (COP)-utilized primers that mismatched the undesired allele within the middle rather than the end of the oligonucleotide (Gibbs et al. 1989; Ruano and Kidd 1989).
Tetra-primer PCR is a method by which two allele-specific amplifications occur in opposite directions (Ye et al. 1992). Tetra-primer PCR and the method of this invention, which we have named Bi-Directional Polymerase Chain Reaction Amplification of Specific Alleles (Bi-PASA), both rely on allele-specific PCR to amplify two alleles simultaneously and in opposite directions. However, the methods differ in the following ways. (1) In tetra-primer PCR, the allele specificity of the inner primers derive from mismatches in the middle of two complementary primers, whereas, in Bi-PASA, the mismatches are at (or near) the 3′ end of the primers. (2) The inner primers in Bi-PASA have short complementary segments and G+C-rich tails to efficiently switch from template-based amplification to self-amplification and to prevent megapriming. (3) Tetra-primer PCR utilizes two annealing conditions of high and low stringency, whereas Bi-PASA utilizes a constant annealing temperature. (4) The inner primers used in tetra-primer PCR are concentrated 35-fold more than the outer primers, whereas in Bi-PASA the primers are of similar concentration.
SUMMARY OF THE INVENTION
This invention is a method for conducting a bi-directional PCR amplification of specific alleles. DNA which may contain one or both of first and second alleles is subjected to a PCR utilizing an outer pair of primers P and Q and an inner pair of primers A and B.
Q is complementary to the sense strand of both alleles in a region downstream of the sequence difference (mismatch) X which distinguishes the alleles. X may be a substitution, deletion or insertion of one or more base pairs. P is complementary to the anti-sense strand of both alleles in a region upstream of X. The terms “upstream” and “downstream” relate to the direction of transcription.
B has a region at its 3′ end which is complementary to the sense strand of the first allele and A has a region at its 3′ end which is complementary to the antisense strand of the second allele. Each of A and B also has a non-complementary G+C-rich tail at its 5′end. X occurs at or near the 3′ end of each of A and B.
DNA which is heterozygous with respect to the two alleles results in amplification of three overlapping sequences, PQ, PB and AQ. PQ, PB and AQ, respectively, stand for the sequences extending from P to Q inclusive, from P to B inclusive, and from A to Q inclusive. DNA which is homozygous with respect to the first allele results in amplification of two overlapping sequences, PQ and PB. DNA which is homozygous with respect to the second allele results in amplification of two overlapping sequences, PQ and AQ.
Following amplification, the sample can be analyzed to determine whether PB and/or AQ is present along with PQ. Preferably, the number of nucleotides separating P from X is sufficiently different from the number of nucleotides separating Q from X that the segments PB and AQ can be distinguished on an agarose gel. However, this is not necessary when other methods of analysis are used.
Bi-PASA provides a rapid, one-tube method for simultaneously differentiating homozygotes and heterozygotes. It is applicable to detecting small deletions and insertions as well as single base changes. The method is particularly useful for determining zygosity in a sample containing a wild-type allele and/or a single-base change mutant and, in general, for determining the zygosity of common sequence changes in which heterozygotes are likely to be common. By using 3 or more inner primers, it can be used to differentiate 3 or more alleles. Bi-PASA can be used to perform population screening, haplotype analysis, patient screening, and carrier testing. It is rapid, reproducible, inexpensive, non-isotopic, and amenable to automation. The method can be conducted simultaneously on 2 or more loci in different tubes, using a uniform annealing temperature.


REFERENCES:
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patent: 5314809 (1994-05-01), Erlich et al.
patent: 5811235 (1998-09-01), Jeffreys
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Gibbs R. A. et al., “Detection of Single DNA Base Differences by Competitive Oligonucleotide Priming”,Nucleic Acids Research,17 (7), 2437-48 (1989).
Newton, C.R. et al., “Analysis of Any Point Mutation in DNA. The Amplification Refactory Mutation System (ARMS)”,Nucleic Acids Research,17 (7), 2503-16 (1989).
Nichols, W.C. et al., “Direct Sequencing of the Gene for Maryland/German Familial Amyloidotic Polyneuropathy Type II and Genotyping by Allele-Specific Enzymatic Amplification”,Genomics,5:535-40 (1989).
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Sarkar G. et al., “Double-Stranded DNA Segments Can Efficiently Prime the Amplification of Human Genomic DNA”,

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