US 11,898,201 B2
Split-cycle and tape amplification
Nicholas Heredia, Mountain House, CA (US); and Dianna Maar, Mountain House, CA (US)
Assigned to Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed by Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed on Jan. 13, 2021, as Appl. No. 17/148,440.
Application 16/361,704 is a division of application No. 15/393,103, filed on Dec. 28, 2016, granted, now 10,280,455, issued on May 7, 2019.
Application 17/148,440 is a continuation of application No. 16/361,704, filed on Mar. 22, 2019, granted, now 10,920,270.
Claims priority of provisional application 62/273,210, filed on Dec. 30, 2015.
Prior Publication US 2021/0207208 A1, Jul. 8, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6865 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/6858 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6848 (2018.01); C12Q 1/6851 (2018.01); C12Q 1/6853 (2018.01)
CPC C12Q 1/6865 (2013.01) [C12Q 1/6816 (2013.01); C12Q 1/6827 (2013.01); C12Q 1/6848 (2013.01); C12Q 1/6851 (2013.01); C12Q 1/6853 (2013.01); C12Q 1/6858 (2013.01)] 9 Claims
OG exemplary drawing
 
1. A plurality of mixture partitions, the individual mixture partitions comprising:
i) a mutation-specific 5′-tailed primer pair, wherein the mutation-specific 5′-tailed primer pair hybridizes to and specifically amplifies target DNA template molecules from a nucleic acid sample that comprise a mutant target sequence, if present, wherein the primers of the mutation-specific 5′-tailed primer pair comprise:
a) a 3′ hybridization region that specifically hybridizes to the mutant target sequence; and
b) a mutation-specific 5′ tail region that does not hybridize to any nucleic acid fragments in the nucleic acid sample;
ii) a wild-type-specific 5′-tailed primer pair, wherein the wild-type-specific 5′-tailed primer pair hybridizes to and specifically amplifies target DNA template molecules comprising a wild-type target sequence, if present, wherein the primers of the wild-type-specific 5′-tailed primer pair comprise:
a) a 3′ hybridization region that specifically hybridizes to the wild-type target sequence; and
b) a wild-type-specific 5′ tail region that does not hybridize to any nucleic acid fragments in the nucleic acid sample, wherein the wild-type-specific 5′ tail region is a different sequence than the mutation-specific 5′ tail region; and
iii) a mutation-specific flanking primer pair, wherein the mutation-specific flanking primer pair hybridizes to and specifically amplifies amplicons comprising the 5′ tail regions of the mutation-specific 5′-tailed primer pair, if present;
iv) a wild-type-specific flanking primer pair, wherein the wild-type-specific flanking primer pair hybridizes to and specifically amplifies amplicons comprising the 5′ tail regions of the wild-type-specific 5′-tailed primer pair, if present; and
v) a thermostable polymerase, wherein
at least some of the mixture partitions of the plurality of mixture partitions contain a target DNA template molecule that comprises a wild-type or mutant target sequence; and
at least some of the mixture partitions of the plurality of mixture partitions do not contain the target DNA template molecule.