Loop-shaped primer used in nucleic acid amplification and the use thereof
Loop-shaped primer used in nucleic acid amplification is an oligonucleotide with 3-20 bases in both 3′ and 5′ ends which can be combined together to form a double-strand under appropriate conditions, resulting in the primer forming a stem-loop structure. The double-stranded structure is opened and the stem-loop structure dissolves when the primer recognizes and hybridizes with the target sequence. If the target sequence is not present the primer can form a stem-loop structure automatically by self-annealing. The primer can comprise a universal tag sequence or not. Together with universal tag sequence primer the primer comprising the universal tag sequence can be used for a second round of amplification. The primer has high specificity and does not form a primer dimmer. The primer is easy to design and is suitable for measuring gene expression and detecting features of nucleic acids such as SNPs and rare mutations.
1. A method of amplifying a nucleic acid, comprising initially denaturing a target sequence containing a mutation sequence, as well as a forward primer and a reverse primer respectively having a target recognizing sequence and complementary sequences on both 3′ end and 5′ end thereof to form a single-loop structure having a blunt end before denaturing, wherein the forward primer further has a mutation recognizing sequence for recognizing the mutation sequence;
annealing the forward primer and the reverse primer with the target sequence;
extending the forward primer and the reverse primer along the target sequence;
denaturing the target sequence, as well as the extended forward and reverse primers, wherein the sequence extended from the forward primer is an object sequence to be amplified by a subsequent polymerase chain reaction (PCR) and wherein the method excludes use of DNA helicase.
2. The method of claim 1 , wherein the mutation recognizing sequence is located on the first base of the 3′ end of the forward primer when the mutation sequence of the target sequence is a point mutation.
3. The method of claim 1 , wherein the mutation recognizing sequence has several contiguous bases in the 3′ end or near the 3′ end when the mutation sequence of the target sequence is a deletion or insertion mutation.
4. The method of claim 1 , wherein a total length of the forward and reverse primers is 20-80 base pairs.
5. The method of claim 1 , wherein a length of the complementary sequences on the 3′ and 5′ ends of the forward and reverse primers is 3-20 base pairs.
6. The method of claim 1 , wherein the forward and the reverse primers comprise at least a non-natural nucleic acid.
7. A method of amplifying a nucleic acid, comprising initially denaturing a target sequence containing a mutation sequence, a tag primer having a tag sequence, as well as a forward primer and a reverse primer respectively having a tag sequence and complementary sequences on both 3′ end and 5′ end thereof to form a single-loop structure having a blunt end before denaturing, wherein the tag primer has a lower melting temperature than the melting temperature of the forward and reverse primers, wherein the forward primer further has a target recognizing sequence and a mutation recognizing sequence for recognizing the mutation sequence, and wherein the reverse primer further has a target combine sequence;
annealing the forward primer and the reverse primer with the target sequence;
extending the forward primer and the reverse primer according to the target sequence;
denaturing the target sequence, as well as the extended forward and reverser primers to obtain a first object sequence extended from the forward primer;
annealing the first object sequence and the reverse primer;
extending the reverse primer along the first object sequence to obtain a second object sequence extended from the reverse primer;
amplifying the second object sequence by the tag primer by a subsequent polymerase chain reaction (PCR) wherein the melting temperature of the complementary sequences when in the single-loop structure is higher than the annealing temperature.
8. The method of claim 7 , wherein the mutation recognizing sequence is located on the first base of the 3′ end of the forward primer when the mutation sequence of the target sequence is a point mutation.
9. The method of claim 7 , wherein the mutation recognizing sequence has several contiguous bases in the 3′ end or near the 3′ end when the mutation sequence of the target sequence is a deletion or insertion mutation.
10. The method of claim 7 , wherein a total length of the forward and reverse primers is 26-80 base pairs.
11. The method of claim 7 , wherein a length of the complementary sequences on the 3′ and 5′ ends of the forward and reverse primers is 3-26 base pairs.
12. The method of claim 7 , wherein the forward and the reverse primers comprise at least a non-natural nucleic acid.