Methods for high specificity whole genome amplification and hybridization
First Claim
1. A method of amplifying a plurality of target sequences from a complex nucleic acid target sample, the method comprising:
- forming an amplification mixture by bringing into contact a set of primers, DNA polymerase, and the complex nucleic acid target sample, wherein the target sample comprises said plurality of target sequences, and wherein the set of primers comprises at least 20 different species of primers, wherein each species of primer is at least 5 bases long, each species of primer has a different sequence and all primers in the set consist of the same two non-complementary bases; and
incubating the amplification mixture under conditions that promote replication of the target sequences, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequences results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.
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Abstract
Methods for amplifying genomic DNA using semi-random primers that consist of different combinations of two non-complementary bases are disclosed. In a preferred aspect all of the primers in the collection are composed entirely of the same two non-complementary bases. In preferred aspects the DNA is amplified using R6, Y6, M6 and K6. The amplification is by a strand displacing polymerase and the amplification product may be hybridized to a high complexity array of probes without further complexity reduction. In some aspects, additives are included in the hybridization to reduce non-specific hybridization. The hybridization pattern obtained is preferably analyzed for allele specific hybridization to determine genotype. The primers in the collection are selected so that there is a minimum of self complementarity between any two primers in the collection, minimizing the occurrence of hybrids between primers.
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Citations
20 Claims
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1. A method of amplifying a plurality of target sequences from a complex nucleic acid target sample, the method comprising:
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forming an amplification mixture by bringing into contact a set of primers, DNA polymerase, and the complex nucleic acid target sample, wherein the target sample comprises said plurality of target sequences, and wherein the set of primers comprises at least 20 different species of primers, wherein each species of primer is at least 5 bases long, each species of primer has a different sequence and all primers in the set consist of the same two non-complementary bases; and
incubating the amplification mixture under conditions that promote replication of the target sequences, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequences results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of amplifying a target nucleic acid sequence, the method comprising:
bringing into contact a set of random ribonucleotide primers, a strand displacing DNA dependent DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence, wherein the target sample is not subjected to denaturing conditions, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand. - View Dependent Claims (11, 12, 13)
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14. A method for determining the genotype of a plurality of polymorphisms in a sample derived from genomic DNA, comprising:
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(a) obtaining a high complexity amplification product by a method comprising amplifying a plurality of nucleic acid sequences in the sample by a complexity amplification method, the complexity amplification method comprising;
mixing the sample with DNA polymerase and a set of at least 20 different sequence primers wherein the primers in the set are at least 6 nucleotides in length and consist of the same two non-complementary bases; and
incubating the mixture under conditions that promote replication of nucleic acid sequences in the sample, wherein replication of the sequences results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand;
(b) fragmenting the high complexity amplification product;
(c) labeling the high complexity amplification product;
(d) hybridizing the high complexity amplification product to a plurality of allele specific probes to obtain a hybridization pattern; and
(e) determining the genotype of a plurality of polymorphisms based on the hybridization pattern. - View Dependent Claims (15, 16, 17, 18, 19, 20)
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Specification