Universal Sanger sequencing from next-gen sequencing amplicons

Patent metadata

Field	Value
Publication number	US-12264357-B2
Application number	US-202117397280-A
Country	US
Kind code	B2
Filing date	Aug 9, 2021
Priority date	Dec 28, 2012
Publication date	Apr 1, 2025
Grant date	Apr 1, 2025

Abstract

Described herein are methods, compositions and kits directed to amplification of nucleic acids suitable for both next generation sequencing (NGS) and a second round of sequencing as validation, such as Sanger sequencing.

First claim

What is claimed is:

1. A method of generating an amplicon for next generation sequencing (NSG) for at least one region of interest comprising:
(a) adding to a sample of DNA:
(i) a first primer set specific for the region of interest consisting of two primers each comprising a 5′ region encoding an adapter and a 3′ region specific for the region of interest, wherein the first primer set binds with a Tm of about 60° C. immediately upstream and downstream of the region of interest; and
(ii) a second primer set consisting of two primers each of which comprises:
(A) a 5′ sequence selected from GTAAAACGACGGCCAGT (SEQ ID NO: 1) or AACAGCTATGACCATG (SEQ ID NO: 2);
(B) a spacer comprising at least 10 nucleotides; and
(C) a 3′ adapter that is substantially identical to the adaptor region,
wherein the adapter has a T m of about 55° C. and the second primer set has a T m of at least 70° C.; and

(b) performing a plurality of PCR cycles with a first annealing temperature and a second annealing temperature.

2. The method of claim 1 , wherein the adaptor comprises a sequence selected from Table 2.

3. The method of claim 1 , wherein the plurality of PCR cycles comprises about 10 cycles at the first annealing temperature and about 30 cycles at the second annealing temperature.

4. The method of claim 3 , wherein the first annealing temperature is about 55° C.

5. The method of claim 3 , wherein the second annealing temperature is between about 60° C. to about 62° C.

6. The method of claim 1 , wherein the plurality of PCR cycles further comprises:
(i) a heating step at about 95° C.;
(ii) an extending step for about 10 cycles of:
(A) about 95° C.;
(B) about 55° C.; and
(C) about 72° C., and

(iii) an extending step for about 30 cycles of:
(A) about 95° C.;
(B) between about 60° C. to about 62° C.; and
(C) about 72° C.

7. The method of claim 6 , wherein the plurality of PCR cycles further comprises an additional extending step at a temperature of about 75° C. for about 15 minutes.

8. The method of claim 1 , wherein the ratio of the first primer set to the second primer set is greater than 1:1.

9. The method of claim 1 , wherein the two primers of the second primer set comprises the sequences selected from SEQ ID NO: 9 or SEQ ID NO: 10.

Assignees

• Quest Diagnostics Invest Llc

Inventors

• Sanders Heather
• Li hai-rong
• Hantash Feras
• Waldman Frederic

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title