SEQUENCE AMPLIFICATION WITH LOOPABLE PRIMERS

• US 20150322504A1
• Filed: 05/27/2015
• Published: 11/12/2015
• Est. Priority Date: 06/20/2007
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: LAO, Kai, STRAUS, Neil
• Granted Patent: US 9,528,147 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2549/119 using nested primers

Abstract

The present disclosure relates to the amplification of target nucleic acid sequences. This can be accomplished via the use of various primers. The use of these primers, as described herein, results in nucleic acid structures that can reduce the amplification of nonspecific hybridization events (such as primer dimerization) while allowing the amplification of the target nucleic acid sequences.

Claims

1. A multiplex amplification reaction mixture comprising:

a) a nucleic acid sample having a plurality of different target nucleic acids; and

b) a mixture of two or more different loopable primers, each loopable primer including

(i) a single-stranded target-specific 3′

region;

(ii) a first stem-forming region;

(iii) a loop-forming region;

(iv) a second stem-forming region; and

(v) a non-complementary single-stranded 5′

tail,

wherein the single-stranded target specific 3′

region includes at least one inosine,

wherein the first and the second stem-forming regions hybridize to each other to form a double-stranded stem region,

wherein the loop-forming region is located between the first and second stem-forming regions, and the loop-forming region contains only adenine residues,

wherein the two or more different loopable primers have non-complementary single-stranded 5′

tails containing different identification sequences, and wherein the stem and the loop, when formed, are configured to allow the single-stranded target specific 3′

region to hybridize to a target nucleic acid without the target nucleic acid hybridizing to the double-stranded stem, the loop-forming region, or the non-complementary 5′

tail.

2. The multiplex amplification reaction mixture of claim 1, wherein the single-stranded target-specific 3′

region comprises a nucleotide sequence that can hybridize to a desired short tandem repeat.

3. The multiplex amplification reaction mixture of claim 1, wherein the single-stranded target-specific 3′

region comprises a nucleotide sequence that can hybridize to a locus selected from one or more of the group consisting of;

TH01, TPOX, CSF1PO, vWA, FGA, D3S1358, D5S818, D7S820, D13S317, D16S539, D8S1179, D18S51, D21S11, D2S1338, D3S1539, D4S2368, D9S930, D10S1239, D14S118, D14S548, D14S562, D16S490, D16S753, D17S1298, D17S1299, D19S253, D19S433, D20S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13AO1, HUMBFXIII, HUMLIPOL, HUMvWFA31, Amelogenin, D12s391, D6S1043, SE33, or any combination thereof.

4. The multiplex amplification reaction mixture of claim 1, wherein the mixture of two or more different loopable primers includes a first loopable primer and a second loopable primer having different sequences that form their respective double-stranded stem regions.

5. The multiplex amplification reaction mixture of claim 1, wherein the nucleic acid sample comprises DNA or RNA.

6. The multiplex amplification reaction mixture of claim 1, further comprising a polymerase or reverse transcriptase.