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.

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 and wherein the universal region is located between the first and second loop forming regions, wherein the universal region is configured so that the nucleic acid sequence can form a self-hybridized structure comprising the universal region, the first loop forming region and the second loop forming region 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: TH01, TPOX, CSF1PO, vWA, FGA, D3S1358, D5S818, D7S820, D13S317, D16S539, D8S1179, D18S51, D21S11, D2S1338, D3S1539, D4S2368, D9S930, D10S1239, D14S118, D14S548, D14S562, D16S490, D16S753, D17S1298, D17S1299, D19S253, D19S433, D 20 S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13AO1, HUMBFXIII, HUMLIPOL, HUMvWFA31, Amelogenin, D12s391, D6S1043, SE33, or any combination thereof. 
     
     
         8 . The nucleic acid sequence of  claim 1 , wherein the noncomplementary region comprises 4-15 thymine nucleic acids. 
     
     
         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;   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.   
     
     
         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 . A method for nucleic acid amplification, said method comprising:
 allowing a 3′ target specific region of a loopable primer to hybridize to a first part of a target nucleic acid sequence, wherein a loop in the loopable primer is configured to allow a 3′ end of the loopable primer to hybridize to a first part of the target nucleic acid sequence without the remainder of the loopable primer annealing to the target nucleic acid sequence, wherein the loopable primer further comprises: a first loop forming region, a universal region, and a second loop forming region, wherein said first and second loop forming regions comprise nucleic acid sequences that hybridize to one another and wherein the universal region is located between the first and second loop forming regions;   extending the loopable primer that is hybridized to the target nucleic acid sequence to form an extended loopable primer;   allowing an additional loopable primer to hybridize to a complementary part of the target nucleic acid sequence on the extended loopable primer; and   extending the loopable primer to form a double-extended loopable primer.   
     
     
         16 . The method of  claim 15 , wherein the 3′ target specific region comprises a degenerate region or a random region. 
     
     
         17 . The method of  claim 15 , further comprising the step of amplifying the double-extended loopable primer through the use of an amplification primer. 
     
     
         18 . The method of  claim 17 , wherein the amplification primer comprises a sequence that is the same as a sequence of the first loop forming region and the universal region. 
     
     
         19 . The method of  claim 17 , wherein the amplification primer consists essentially of the universal region. 
     
     
         20 . The method of  claim 15 , wherein the 3′ target specific region comprises a sequence that allows for the amplification of a short tandem repeat within the resulting extended loopable primer. 
     
     
         21 . The method of  claim 15 , further comprising the step of self-hybridizing the double-extended loopable primer via at least the universal region and a sequence that is complementary to the universal region. 
     
     
         22 . The method of  claim 21 , wherein the self-hybridization occurs via the first loop forming region, the universal region and the second loop forming region. 
     
     
         23 . The method of  claim 15 , wherein only one loopable primer sequence is used for the hybridization to the target nucleic acid sequence. 
     
     
         24 . The method of  claim 15 , wherein loopable primers having different sequences are used for the hybridization to the target nucleic acid sequence. 
     
     
         25 . The method of  claim 24 , wherein the loopable primers have a sequence between a first 3′ target specific region and a region that is complementary to a second 3′ target specific region. 
     
     
         26 . The method of  claim 25 , wherein at least 10 different loopable primers are added prior to the hybridization and extension step, wherein each of the 10 different loopable primers comprise a different 3′ target specific region. 
     
     
         27 . The method of  claim 15 , further comprising the step of self-hybridizing the double-extended loopable primer via the universal region and a sequence that is complementary to the universal region, wherein the self-hybridized double-extended loopable primer has an insert section between the two sections that are hybridized to one another. 
     
     
         28 . The method of  claim 27 , wherein the insert section is large enough to allow amplification of a part of the target nucleic acid sequence within the loop. 
     
     
         29 . The method of  claim 27 , wherein the insert section is short enough to reduce the likelihood that amplification will occur within the loop. 
     
     
         30 . The method of  claim 27 , wherein the self-hybridization occurs at a rate that is slower than the self-hybridization rate of a primer-dimer self-hybridizing. 
     
     
         31 . The method of  claim 27 , further comprising the step of PCR amplifying a part of the insert section. 
     
     
         32 . The method of  claim 27 , further comprising the step of selectively amplifying a double-extended loopable primer that comprises a target nucleic acid sequence over a self-hybridized double-extended loopable primer that lacks a target nucleic acid sequence, apart from the 3′ target specific region. 
     
     
         33 . The method of  claim 27 , further comprising the step of amplifying at least a section of the insert section by using at least one insert amplification primer. 
     
     
         34 . The method of  claim 33 , further comprising the step of amplifying at least a section of the insert section by using at least a second insert amplification primer. 
     
     
         35 . The method of  claim 33 , wherein the insert amplification primer hybridizes to a specific nucleic acid sequence, the presence of which is desired to be amplified. 
     
     
         36 . The method of  claim 33 , wherein the insert amplification primer comprises a nucleotide sequence that can amplify a locus selected from one or more of the group consisting of: TH01, TPOX, CSF1PO, vWA, FGA, D3S1358, D5S818, D7S820, D13S317, D16S539, D8S1179, D18S51, D21S11, D2S1338, D3S1539, D4S2368, D9S930, D10S1239, D14S118, D14S548, D14S562, D16S490, D16S753, D17S1298, D17S1299, D19S253, D19S433, D 20 S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13AO1, HUMBFXIII, HUMLIPOL, HUMvWFA31, Amelogenin, D12s391, D6S1043, SE33, or any combination thereof. 
     
     
         37 . The method of  claim 33 , wherein at least 10 insert amplification primers are used for the insert section amplification, wherein the double-extended loopable primer is contained within a solution, wherein the solution is divided into at least 10 parts, each part containing at least one double-extended loopable primer, and wherein each one of the at least 10 different insert amplification primers is added to a different part, and wherein the step of amplifying occurs separately in each part. 
     
     
         38 . The method of  claim 15 , further comprising the step of removing random primers. 
     
     
         39 . The method of  claim 38 , wherein the removal of the random primers is achieved by a digestion of unpaired primers. 
     
     
         40 . The method of  claim 38 , wherein the removal of the random primer is performed with exonuclease I. 
     
     
         41 . The method of  claim 38 , wherein the target nucleic acid sequence is a gDNA sequence. 
     
     
         42 . The method of  claim 15 , wherein said random region has a frequency that is sufficiently common in a sample to allow for near or complete whole genome amplification. 
     
     
         43 . The method of  claim 15 , further comprising the steps of:
 amplifying the double-extended loopable primer through the use of an amplification primer that hybridizes to the complement of the loopable primer;   removing random primers;   self-hybridizing two ends of a first double-extended loopable primer together;   not self-hybridizing two ends of a second double-extended loopable primer together, wherein the second self-hybridized double-extended loopable primer has an insert section between the two sections that are hybridized to one another; and   selectively amplifying a part of an insert section of the second double-extended loopable primer over the first double-extended loopable primer.   
     
     
         44 . The method of  claim 43 , wherein the steps are performed in the order in which they are listed in  claim 43 . 
     
     
         45 . The method of  claim 15 , further comprising the steps of:
 initially performing a reverse transcription reaction on a sample that comprises RNA, wherein the product of the reverse transcription reaction is a cDNA that comprises the target nucleic acid sequence;   amplifying the double-extended loopable primer once the double-extended loopable primer has been formed;   removing random primers; and   selectively amplifying at least a part of an insert section of a first double-extended loopable primer over a second double-extended loopable primer, wherein two ends of the first double-extended loopable primer are not hybridized together, and wherein two ends of the second double-extended loopable primer are hybridized together.   
     
     
         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 and 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 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.