US 9,809,845 B2
Methods and reagents for amplifying nucleic acids
Jothikumar Narayanan, Atlanta, GA (US); Prithiviraj Jothikumar, Atlanta, GA (US); and Vincent Hill, Decatur, GA (US)
Assigned to The United States of America, as represented by the Secretary, Department of Health and Human Services, Washington, DC (US)
Appl. No. 14/419,641
Filed by Jothikumar Narayanan, Atlanta, GA (US); Prithiviraj Jothikumar, Atlanta, GA (US); and Vincent Hill, Decatur, GA (US)
PCT Filed Aug. 6, 2012, PCT No. PCT/US2012/049784
§ 371(c)(1), (2), (4) Date Feb. 4, 2015,
PCT Pub. No. WO2014/025337, PCT Pub. Date Feb. 13, 2014.
Prior Publication US 2015/0159205 A1, Jun. 11, 2015
Int. Cl. C12P 19/34 (2006.01); C12Q 1/68 (2006.01); C12Q 1/70 (2006.01)
CPC C12Q 1/6844 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/689 (2013.01); C12Q 1/6888 (2013.01); C12Q 1/701 (2013.01); C12Q 2600/158 (2013.01)] 29 Claims
 
1. A method of amplifying a target nucleic acid molecule comprising from 5′ to 3′, a 5′-end region, a center region, and a 3′-end region, wherein each region is non-overlapping and comprises at least 10 consecutive nucleotides, comprising:
contacting the target nucleic acid molecule under conditions sufficient for isothermal amplification with an effective amount of:
a forward turn-back primer comprising a first segment comprising at least 10 consecutive nucleotides capable of hybridizing to the center region and a second segment comprising at least 10 consecutive nucleotides capable of hybridizing to the complement of the 5′-end region, wherein the first segment is 5′ to the second segment; and
a reverse turn-back primer comprising a first segment comprising at least 10 consecutive nucleotides capable of hybridizing to the complement of the center region and a second segment comprising at least 10 consecutive nucleotides capable of hybridizing to the 3′-end region, wherein the first segment is 5′ to the second segment; and
amplifying the target nucleic acid molecule using isothermal amplification.