Hybrid multi-step nucleic acid amplification
Improved methods for amplifying target nucleic acid sequences are provided by 1) first amplifying the number of copies of target nucleic acid sequences in a sample by a first nucleic acid amplification method, and then 2) applying a second nucleic amplification method to the amplified sample, or aliquot thereof, further amplifying the number of copies of target sequences. In embodiments, a first nucleic acid amplification method is a thermocycling method, and a second nucleic acid amplification method is an isothermal method.
1. A method for amplifying a polynucleotide template, the method comprising:
A) generating multiple copies of a polynucleotide template in a polymerase chain reaction (PCR) amplification reaction mixture, wherein the sample is derived from capillary whole blood wherein the PCR amplification reaction mixture comprises a first PCR amplification reaction primer and a second PCR amplification reaction primer, wherein in the PCR amplification reaction mixture, the first PCR amplification reaction primer anneals to the polynucleotide template and the second PCR amplification reaction primer anneals to a polynucleotide which is complementary to the polynucleotide template, and wherein in the PCR amplification reaction mixture, multiple copies of a PCR amplification reaction product are formed, wherein the PCR amplification reaction product is a double-stranded nucleic acid molecule comprising a first strand and a second strand, and wherein a first strand of the PCR amplification reaction product is a copy of the polynucleotide template;
B) incubating copies of the polynucleotide template in a non-thermocycling reaction mixture comprising a non-thermocycling reaction first primer and a non-thermocycling reaction second primer, wherein:
the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion;
the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the polynucleotide template; and
the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to a sequence in the PCR amplification reaction product second strand which is complementary to the second portion of the polynucleotide template, the first region of the second primer is complementary to the first region of the first primer, and the first region of the second primer is complementary to the third portion of the polynucleotide template.
2. The method of claim 1 , wherein the first portion and second portion of the polynucleotide template are each between 6 and 30 nucleotides in length.
3. The method of claim 1 , wherein the third portion of the polynucleotide template is between 4 and 14 nucleotides in length.
4. A method for amplifying a polynucleotide template, the method comprising:
A) generating multiple copies of a polynucleotide template in a polymerase chain reaction (PCR) amplification reaction mixture, wherein the sample is derived from capillary whole blood wherein the PCR amplification reaction mixture comprises a first PCR amplification reaction primer and a second PCR amplification reaction primer, wherein in the PCR amplification reaction mixture, the first PCR amplification reaction primer anneals to the polynucleotide template and the second PCR amplification reaction primer anneals to a polynucleotide which is complementary to the polynucleotide template, and wherein in the PCR amplification reaction mixture, multiple copies of a PCR amplification reaction product are formed, wherein the PCR amplification reaction product is a double-stranded nucleic acid molecule comprising a first strand and a second strand, and wherein a first strand of the PCR amplification reaction product is a copy of the polynucleotide template; B) incubating copies of the polynucleotide template in a non-thermocycling reaction mixture comprising a non-thermocycling reaction first primer and a non-thermocycling reaction second primer, wherein:
the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion;
the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the polynucleotide template; and the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to a sequence in the PCR amplification reaction product second strand which is complementary to the second portion of the polynucleotide template, the first region of the second primer is complementary to the first region of the first primer, and the first region of the second primer is complementary to the third portion of the polynucleotide template;
wherein the number of copies of the polynucleotide template in the non-thermocycling reaction mixture is increased at least 10-fold within 60 minutes of initiation of the method.
5. A method for amplifying a polynucleotide template, the method comprising:
A) generating multiple copies of a polynucleotide template in a polymerase chain reaction (PCR) amplification reaction mixture, wherein the sample is derived from capillary whole blood wherein the PCR amplification reaction mixture comprises a first PCR amplification reaction primer and a second PCR amplification reaction primer, wherein in the PCR amplification reaction mixture, the first PCR amplification reaction primer anneals to the polynucleotide template and the second PCR amplification reaction primer anneals to a polynucleotide which is complementary to the polynucleotide template, and wherein in the PCR amplification reaction mixture, multiple copies of a PCR amplification reaction product are formed, wherein the PCR amplification reaction product is a double-stranded nucleic acid molecule comprising a first strand and a second strand, and wherein a first strand of the PCR amplification reaction product is a copy of the polynucleotide template; B) incubating copies of the polynucleotide template in a non-thermocycling reaction mixture comprising a non-thermocycling reaction first primer and a non-thermocycling reaction second primer, wherein:
the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion;
the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the polynucleotide template; and the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to a sequence in the PCR amplification reaction product second strand which is complementary to the second portion of the polynucleotide template, the first region of the second primer is complementary to the first region of the first primer, and the first region of the second primer is complementary to the third portion of the polynucleotide template;
wherein a concatemer strand comprising at least three copies of the polynucleotide template is generated during the incubation of the non-thermocycling reaction mixture.