Sequence amplification with loopable primers

Abstract

The present disclosure relates to the amplification of target nucleic acid sequences. This can be accomplished via the use of various primers. The use of these primers, as described herein, results in nucleic acid structures that can reduce the amplification of nonspecific hybridization events (such as primer dimerization) while allowing the amplification of the target nucleic acid sequences.

What is claimed is: 
     
         1 . A nucleic acid sequence comprising:
 a 3′ target specific region;   a first loop forming region;   a universal region; and   a second loop forming region,   wherein said first and second loop forming regions comprise a set of nucleic acid sequences that are configured to hybridize to one another to form a stem and loop,   wherein the universal region is located between the first and second loop forming regions,   wherein the stem and the loop, when formed, are configured to allow the 3′ target specific region to hybridize to a target nucleic acid without the remainder of the nucleic acid sequence annealing to the target nucleic acid, and   wherein the universal region is configured to form a self-hybridized structure on subsequent amplifications.   
     
     
         2 . The nucleic acid sequence of  claim 1 , wherein the 3′ target specific region comprises a degenerate sequence. 
     
     
         3 . The nucleic acid sequence of  claim 1 , further comprising a noncomplementary region located in the nucleic acid sequence between the first universal region and the second loop forming region. 
     
     
         4 . The nucleic acid sequence of  claim 1 , further comprising a foreign insert section of DNA. 
     
     
         5 . The nucleic acid sequence of  claim 4 , wherein the foreign insert section of DNA is longer than the length of the 3′ target specific region. 
     
     
         6 . The nucleic acid sequence of  claim 1 , wherein the 3′ target specific region comprises a nucleotide sequence that can amplify a desired short tandem repeat. 
     
     
         7 . The nucleic acid sequence of  claim 1 , wherein the 3′ target specific region comprises a nucleotide sequence that can amplify a locus selected from one or more of the group consisting of: THOI, TPDX, CSFIPO, vWA, FGA, D3S1358, D5S8I8, D7S820, D13S3I7, DI6S539, D8S1179, DI8S5I, D2IS11, D2S1338, D3SI539, D4S2368, D9S930, DIOSI239, DI4S118, DI4S548, DI4S562, DI6S490, DI6S753, DI7SI298, DI7SI299, DI9S253, DI9S433, D20S48I, D22S683, HUMCSFIPO, HUMTPDX, HUMTHOI,HUMF13A01, HUMBFXIII, HUMLIPOL, HUMvWFA31, Amelogenin, D12s391, D6S1043, SE33, or any combination thereof. 
     
     
         8 . The nucleic acid sequence of  claim 3 , wherein the noncomplementary region comprises 4-15 thymine nucleotides. 
     
     
         9 . The nucleic acid sequence of  claim 8 , wherein the noncomplementary region consists essentially of 7-15 thymine nucleotides. 
     
     
         10 . A self-hybridizable DNA structure in a PCR amplification mixture, said self-hybridizable DNA sequence comprising:
 a first loop forming region;   a universal region connected to the first loop forming region;   a second loop forming region connected to the universal region, wherein the first loop forming region and the second loop forming region are complementary to each other;   a first 3′ target specific region connected to the second loop forming region;   a sequence that is complementary to a second 3′ target specific region, wherein the sequence that is complementary to the second 3′ target specific region is part of a same nucleic acid strand as the first 3′ target specific region;   a sequence that is complementary to the second loop forming region, wherein the sequence that is complementary to the second loop forming region is connected to the sequence that is complementary to the second 3′ target specific region;   a sequence that is complementary to the universal region, wherein the a sequence that is complementary to the universal region is connected to the sequence that is complementary to the second loop forming region; and   a sequence that is complementary to the first loop forming region, wherein the sequence that is complementary to the first loop forming region is connected to the sequence that is complementary to the universal region,   wherein the self-hybridizable DNA structure is configured to form a stem and a loop structure, wherein the stem is formed by hybridization of the sequence comprising the first loop forming region, the universal region and the second loop forming region to the comprising their respective complementary sequences.   
     
     
         11 . The self-hybridized DNA structure of  claim 10 , wherein there is no cDNA located within or between the 3′ target specific region and the sequence that is complementary to the second 3′ target specific region. 
     
     
         12 . The self-hybridized DNA structure of  claim 10 , wherein the amount of cDNA located between the 3′ target specific region and the sequence that is complementary to the second 3′ target specific region is less than 500 base pairs. 
     
     
         13 . The self-hybridized DNA structure of  claim 10 , wherein the amount of cDNA
 located between the 3′ target specific region and the sequence that is complementary to the second 3′ target specific region is less than 100 base pairs.   
     
     
         14 . The self-hybridized DNA structure of  claim 10 , wherein the amount of cDNA located between the 3′ target specific region and the sequence that is complementary to the second 3′ target specific region is less than 50 base pairs. 
     
     
         15 - 45 . (canceled) 
     
     
         46 . A nucleic acid sequence comprising:
 a 3′ target specific region;   a first loop forming region;   a universal region;   a noncomplementary region; and   a second loop forming region, wherein said first and second loop forming regions comprise a set of nucleic acid sequences that are configured to hybridize to one another to form a stem and a loop,   wherein the universal region is located between the first and second loop forming regions,   wherein the noncomplementary region is located between the first and second loop forming regions,   wherein the stem and the loop, when formed, are configured to allow the 3′ target specific region to hybridize to a target nucleic acid without the remainder of the loopable primer annealing to the target nucleic acid, and   wherein the universal region is configured so that a sequence associated with a target sequence that has been amplified by the nucleic acid sequence can form a self-hybridized structure comprising the universal region, the noncomplementary region, the first loop forming region, and the second loop forming region on subsequent amplifications, wherein said nucleic acid is created by deliberately selecting the sequence of the first loop forming region so that it hybridizes to the second loop forming region, and wherein said nucleic acid is created by deliberately selecting a universal region.   
     
     
         47 . The nucleic acids sequence of  claim 2  wherein the 3′ target specific region comprises a degenerate end. 
     
     
         48 . The nucleic acids sequence of  claim 1  wherein the 3′ target specific region comprises a random region. 
     
     
         49 . The nucleic acids sequence of  claim 48  wherein the 3′ target specific region is a random region.