Method of double allele specific PCR for SNP microarray

• US 10,457,989 B2
• Filed: 05/24/2016
• Issued: 10/29/2019
• Est. Priority Date: 12/28/2015
• Status: Active Grant

• Current Assignee: Phalanx Biotech Group, Inc.
• Original Assignee: Phalanx Biotech Group, Inc.
• Inventors: Lin, Shang-Chi
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/162,907
• Publication Number:
  US 20170183729A1
• Time in Patent Office: 1,253 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• 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
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

The present invention provides a method of double allele specific polymerase chain reaction (PCR) for single nucleotide polymorphism (SNP) microarray, the method provides the allele specific site as the 3′ terminal nucleotide of forward and reverse primers, it does not require a primer having specific nucleotides. The method of the present invention is easily to design the primer based on flanking region of the allele specific site, and to perform multiplex SNP PCR applying with an interchelating agent, then to detect by a SNP microarray.

Claims

1. A method of double allele specific polymerase chain reaction (PCR) for single nucleotide polymorphism (SNP) microarray, comprising:

(a) amplifying a nucleic acid sample by multiplex SNP PCR using at least a primer set which recognizes a single nucleotide polymorphism (SNP) to obtain an amplified product;

wherein an interchelating agent is added into PCR to inhibit non-specific amplification, and the interchelating agent is N′

,N′

-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine; and

(b) contacting the amplified product with a SNP microarray fixed probe suitable for determining the identity of SNP to genotype SNP,

wherein the primer set contains at least four allele specific primers, which at least two allele specific primers bound with a sense strand of the nucleic acid sample, and 3′

terminal nucleotides of the two allele specific primers are complementary to an allele specific site; and

at least two allele specific primers bound with an antisense strand of the nucleic acid sample, and 3′

terminal nucleotides of the two allele specific primers are complementary to the allele specific site;

wherein the allele specific primers have melting temperature (Tm) values, and the Tm values have maximum temperature difference of 2.8°

C. in single PCR.

2. The method according to claim 1, wherein the nucleic acid sample is obtained from blood, serum, plasma, body fluids or tissues.

3. The method according to claim 1, wherein the SNP microarray is detected by a fluorescent array imaging reader.

4. The method according to claim 1, wherein a 5′

terminal end of the allele specific primer further comprises a modified label.

5. The method according to claim 1, further comprising adding a specific modified base during the multiplex SNP PCR amplification.

6. A method of double allele specific polymerase chain reaction (PCR) for single nucleotide polymorphism (SNP) microarray by identifying a modified label, comprising:

(a) amplifying a nucleic acid sample by multiplex SNP PCR using at least a primer set which recognizes a single nucleotide polymorphism (SNP) to obtain a first amplified product;

wherein an interchelating agent is added into PCR to inhibit non-specific amplification, and the interchelating agent is N′

,N′

-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine;

(b) amplifying the first amplified product using a universal PCR primer having a modified label at 5′

terminal end to obtain a second amplified product; and

(c) contacting the second amplified product with a SNP microarray fixed probe suitable for determining the identity of SNP to genotype SNP,

wherein the sequence of the primer set contains two regions, which 5′

terminal region of the allele specific primer is a universal PCR primer sequence, and 3′

terminal region of the allele specific primer is an allele specific primer region; and

wherein the primer set contains at least four allele specific primers, which at least two allele specific primers bound with a sense strand of the nucleic acid sample, and the 3′

terminal region of the two allele specific primers are complementary to an allele specific site; and

at least two allele specific primers bound with an antisense strand of the nucleic acid sample, and the 3′

terminal region of the two allele specific primers are complementary to the allele specific site;

wherein the allele specific primers have melting temperature (Tm) values, and the Tm values have maximum temperature difference of 2.8°

C. in single PCR.

7. The method according to claim 6, wherein the modified label is fluorescence, biotin, digoxigenin (DIG) or oligonucleotide sequence.

8. The method according to claim 6, wherein the nucleic acid sample is obtained from blood, serum, plasma, body fluids or tissues.

9. The method according to claim 6, wherein the allele specific primers further comprise a barcode sequence between the universal PCR primer sequence and the allele specific primer region;

and the probe of step (c) comprises a sequence complementary to the barcode sequence.

10. The method according to claim 6, wherein the SNP microarray is detected by a fluorescent array imaging reader.