Multiple displacement amplification

• US 6,280,949 B1
• Filed: 09/17/1999
• Issued: 08/28/2001
• Est. Priority Date: 10/08/1997
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Lizardi, Paul M.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/397,915
• Time in Patent Office: 711 Days
• Field of Search: 435/6, 435/91.2, 435/91.52, 435/91.51, 435/91.1, 536/24.3
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The method is based on stand displacement replication of the nucleic acid sequences of interest by multiple primers. In one preferred form of the method, referred to as multiple strand displacement amplification, two sets of primers are used, a right set and a left set. The primers in the right set are complementary to one strand of the nucleic acid molecule to be amplified and the primers in the left set are complementary to the opposite strand. The 5′ end of primers in both sets are distal to the nucleic acid sequence of interest when the primers have hybridized to the nucleic acid sequence molecule to be amplified. Amplification proceeds by replication initiated at each primer and continuing through the nucleic acid sequence of interest. A key feature of this method is the displacement of intervening primers during replication by the polymerase. In another preferred form of the method, referred to as whole genome strand displacement amplification, a random set of primers is used to randomly prime a sample of genomic nucleic acid (or another sample of nucleic acid of high complexity). By choosing a set of primers which are sufficiently random, the primers in the set will be collectively, and randomly, complementary to nucleic acid sequences distributed throughout nucleic acid in the sample. Amplification proceeds by replication with a highly processive polymerase initiated at each primer and continuing until spontaneous termination. A key feature of this method is the displacement of intervening primers during replication by the polymerase. In this way, multiple overlapping copies of the entire genome to be synthesized in a short time.

Claims

1. A method of amplifying a target nucleic acid sequence, the method comprising,

(a) mixing a set of primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the primers and the target sequence in the primer-target sample mixture,

(b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence,

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,

wherein the target sequence is a nucleic acid sample of substantial complexity, and wherein the set of primers comprises primers having random nucleotide sequences,

wherein each primer comprises a constant portion and a random portion, wherein the constant portion of each primer has the same nucleotide sequence and the random portion of each primer has a random nucleotide sequence.

2. A method of amplifying a target nucleic acid sequence, the method comprising,

(a) mixing a set of primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the primers and the target sequence in the primer-target sample mixture,

(b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence,

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,

wherein the conditions that promote replication of the target sequence do not involve thermal cycling, and

wherein the target sequence is concatenated DNA.

3. The method of claim 2 wherein the concatenated DNA is concatenated with linkers.

4. The method of claim 3 wherein each linker comprises a primer complement portion, wherein each primer comprises a complementary portion, wherein the complementary portion of each primer is complementary to the complementary portion of the linkers.

5. The method of claim 2 wherein the set of primers comprises primers having random nucleotide sequences.

6. The method of claim 5 wherein each primer comprises a constant portion and a random portion, wherein the constant portion of each primer has the same nucleotide sequence and the random portion of each primer has a random nucleotide sequence.

7. The method of claim 2 wherein the concatenated DNA is formed by ligating DNA fragments together.

8. The method of claim 7 wherein the DNA fragments are cDNA made from mRNA.

9. The method of claim 8 wherein the mRNA comprises a mixture of mRNA isolated from cells.