Methods for multiplex PCR

• US 10,316,359 B2
• Filed: 08/31/2016
• Issued: 06/11/2019
• Est. Priority Date: 01/31/2014
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Swift Biosciences, Inc. (Integrated DNA Technologies, Inc.)
• Inventors: Makarov, Vladimir, Laliberte, Julie
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/252,397
• Publication Number:
  US 20160362726A1
• Time in Patent Office: 1,014 Days
• Field of Search: None
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

Methods for the preparation of PCR reaction mixtures and for performing multiplex PCR amplification which limit the production of non-target amplicons are provided. Production of non-target amplicons is limited because the non-target amplicons have complementary 5′ and 3′ ends as a result of the target-specific primer designs such that the non-target amplicons can form stable secondary structure during the annealing step.

Claims

1. A method of multiplex PCR amplification of a target nucleic acid substrate comprising the steps of:

(i) combining a plurality of target-specific primers with the target nucleic acid substrate to yield a single polymerase chain reaction (PCR) reaction mixture, wherein the plurality of target-specific primers comprise a first forward primer, a second forward primer, a first reverse primer and a second reverse primer, wherein each of the first and second forward and reverse primers comprise a 3′

complementary sequence that is complementary to the target nucleic acid substrate and a 5′

noncomplementary sequence that is not complementary to the target nucleic acid substrate, wherein the 3′

complementary sequence for each of the first and second forward and reverse primers is different;

(ii) subjecting the PCR reaction mixture to a multiplex polymerase chain reaction thereby generating at least three amplicons, wherein the at least three amplicons comprise a first amplicon produced by the first forward primer and the first reverse primer, a second amplicon produced by the second forward primer and the second reverse primer, and a third amplicon produced by the second forward primer and the first reverse primer, wherein at least a portion of the 5′

noncomplementary sequence of the second forward primer and the first reverse primer is the same such that each strand of the third amplicon comprises a 3′

end and a 5′

end that are complementary to each other, wherein the third amplicon possesses overlapping sequence with the first and second amplicons, wherein the first amplicon possesses overlapping sequence with the second amplicon, wherein when the third amplicon is denatured, each strand of the third amplicon forms a secondary structure as a result of the 3′

end being complementary to the 5′

end, and wherein the secondary structure is stable during a primer annealing step of the multiplex polymerase chain reaction.

2. The method of claim 1, wherein the at least three amplicons further comprise a fourth amplicon produced by the first forward primer and the second reverse primer, and wherein the fourth amplicon overlaps the first, second, and third amplicon.

5. The method of claim 1, wherein the entire 5′

noncomplementary sequence of the second forward primer and the first reverse primer is the same.

3. A method for preparing a PCR reaction mixture for multiplex PCR amplification of a target nucleic acid substrate comprising:

combining a plurality of target-specific primers with the target nucleic acid substrate to yield a single polymerase chain reaction (PCR) reaction mixture, wherein the plurality of target-specific primers comprise a first forward primer, a second forward primer, a first reverse primer and a second reverse primer, wherein each of the first and second forward and reverse primers comprise a 3′

complementary sequence that is complementary to the target nucleic acid substrate and a 5′

noncomplementary sequence that is not complementary to the target nucleic acid substrate, wherein the 3′

complementary sequence for each of the first and second forward and reverse primers is different, and wherein the target specific primers are designed in a manner capable of producing at least three amplicons of the target nucleic acid substrate, wherein the at least three amplicons comprise a first amplicon produced by the first forward primer and first reverse primer, a second amplicon produced by the second forward primer and the second reverse primer, and a third amplicon produced by the second forward primer and the first reverse primer, wherein at least a portion of the 5′

noncomplementary sequence of the second forward primer and the first reverse primer is the same such that each strand of the third amplicon comprises a 3′

end and a 5′

end that are complementary to each other, wherein the third amplicon possesses overlapping sequence with the first and second amplicons, wherein the first amplicon possesses overlapping sequences with the second amplicon, wherein when the third amplicon is denatured, each strand of the third amplicon forms a secondary structure as a result of the 3′

end being complementary to the 5′

end, and wherein the secondary structure is stable during a primer annealing step of a multiplex polymerase chain reaction.

4. The method of claim 3, wherein the at least three amplicons further comprise a fourth amplicon produced by the first forward primer and the second reverse primer, and wherein the fourth amplicon overlaps the first, second, and third amplicon.

6. The method of claim 3, wherein the entire 5′

noncomplementary sequence of the second forward primer and the first reverse primer is the same.