Sequence amplification with linear primers

Abstract

The present disclosure relates to the amplification of target nucleic acid sequences for various sequencing and/or identification techniques. The use of these primers, as described herein, allows for the reduction in the amplification of nonspecific hybridization events (such as primer dimerization) while allowing for the amplification of the target nucleic acid sequences.

1 . A method of amplifying a nucleic acid sequence in a parallel sequencing technique, said method comprising:
 hybridizing a 3′ target specific region of a first linear primer to a first part of a target nucleic acid sequence, wherein the first linear primer comprises a universal priming region that is distinct from the 3′ target specific region, and wherein the 3′ target specific region is a random region that is less than 6 nucleotides in length;   extending the first linear primer that is hybridized to the target nucleic acid sequence to form an extended linear primer;   hybridizing a second linear primer to a complementary part of the target nucleic acid sequence of the extended linear primer, wherein the second linear primer comprises a same universal priming region as the first linear primer;   extending the second linear primer to form a double-extended linear primer;   amplifying the double-extended linear primer using an amplification primer, wherein the amplification primer comprises a sequence that is the same as a sequence of the universal priming region, wherein a double-extended linear primer comprising a first insert section is selectively amplified over a double-extended linear primer comprising a second insert section;   adding a third primer that comprises a sequence that is complementary to a sequence within the first insert section;   performing an amplification reaction using the third primer to amplify the target nucleic acid sequence within the first insert section of the double-extended linear primer, wherein the double-extended linear primer comprising the first insert has its insert section selectively amplified compared to the double-extended linear primer comprising the second insert, wherein the first insert section is larger than the second insert section; and   performing a parallel sequencing reaction on an amplified product from the amplification reaction using the third primer.   
     
     
         2 . The method of  claim 1 , wherein the random region is 4 nucleotides in length. 
     
     
         3 . The method of  claim 1 , wherein the parallel sequencing technique is supported oligo ligation detection. 
     
     
         4 . The method of  claim 1 , wherein a fragment size selection step is not carried out on the sample prior to the steps recited in  claim 1 . 
     
     
         5 . The method of  claim 1 , wherein the self-hybridized structure has a melting temperature in excess of 50° C. 
     
     
         6 . The method of  claim 1 , further comprising selectively amplifying the self-hybridized double-extended linear primer that comprises a target nucleic acid sequence over a self-hybridized double-extended linear primer that lacks a target nucleic acid sequence, apart from the 3′ target specific region. 
     
     
         7 . The method of  claim 1 , further comprising the step of removing random primers. 
     
     
         8 . The method of  claim 1 , wherein the removal of the random primers is achieved by a digestion of unpaired primers. 
     
     
         9 . The method of  claim 1 , wherein the target nucleic acid sequence is a gDNA sequence. 
     
     
         10 . The method of  claim 1 , wherein said random region has a frequency that is sufficiently common in a sample to allow for whole genome amplification. 
     
     
         11 . The method of  claim 1 , wherein the steps are performed in the order in which they are listed in  claim 1 . 
     
     
         12 . The method of  claim 1 , wherein the target nucleic acid sequence is from a sample that comprises fewer than 100 cells. 
     
     
         13 . The method of  claim 1 , wherein the target nucleic acid sequence is from a sample that comprises fewer than 10 cells. 
     
     
         14 . The method of  claim 1 , wherein the target nucleic acid sequence is from a single cell. 
     
     
         15 . The method of  claim 1 , wherein the random region comprises adenine, guanine, and at least one additional, different type of nucleotide. 
     
     
         16 . The method of  claim 1 , wherein the random region comprises at least one thymine and at least one cytosine. 
     
     
         17 . The method of  claim 1 , wherein the random region comprises at least two thymines. 
     
     
         18 . The method of  claim 1 , wherein the random region comprises at least two cytosines. 
     
     
         19 . The method of  claim 1 , further comprising 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. 
     
     
         20 . The method of  claim 1 , wherein the first linear primer further comprises a noncomplementary region that is separate from the 3′ target specific region and the universal priming region. 
     
     
         21 . The method of  claim 20 , wherein the second linear primer comprises a same noncomplementary region as the first linear primer. 
     
     
         22 . The method of  claim 21 , wherein the first and second linear primers are identical primers. 
     
     
         23 . The method of  claim 1 , wherein the random region comprises at least one cytosine or thymine. 
     
     
         24 . The method of  claim 1 , wherein the volume of a liquid in which at least one of the amplification steps is greater than 60 nl, wherein the random region comprises at least one cytosine or at least one thymine, and wherein the parallel sequencing reaction comprises a supported oligo ligation detection technique. 
     
     
         25 . The method of  claim 1 , wherein the first insert section is large enough to allow amplification of the first insert section when the first linear primer within the double-extended linear primer is hybridized to a sequence that is complementary to the first linear primer sequence that is also within the double-extended linear primer, thereby allowing the double-extended linear primer comprising the first insert section to be selectively amplified over a double-extended linear primer comprising the second insert section. 
     
     
         26 . The method of  claim 25 , wherein the first insert section is at least 100 nucleotides in length. 
     
     
         27 . The method of  claim 26 , wherein the first insert section is at least 200 nucleotides in length. 
     
     
         28 . The method of  claim 1 , wherein the double-extended linear primer comprising the second insert section allows the first linear primer to hybridize to a sequence in the double-extended linear primer that is complementary to the first linear primer faster than the amount of time required for the first linear primer to hybridize to a sequence in the double-extended linear primer that is complementary to the first linear primer in the first insert section, thereby allowing the double-extended linear primer comprising the first insert section to be selectively amplified over a double-extended linear primer comprising the second insert section. 
     
     
         29 . The method of  claim 1 , further comprising providing a fourth primer that allows for amplification of the insert section. 
     
     
         30 . The method of  claim 29 , wherein the fourth primer consists of the universal priming region. 
     
     
         31 . The method of  claim 1 , wherein the amplification primer consists essentially of the universal priming region. 
     
     
         32 . The method of  claim 1 , wherein the double extended linear primer comprising the first insert section is not in a looped configuration when the amplification reaction using the third primer to amplify the target nucleic acid sequence occurs. 
     
     
         33 . The method of  claim 32 , wherein the double extended linear primer comprising the second insert section is in a looped configuration when the amplification reaction using the third primer to amplify the target nucleic acid sequence occurs. 
     
     
         34 . The method of  claim 1 , wherein the double extended linear primer comprising the second insert section is in a looped configuration when the amplification reaction using the third to amplify the target nucleic acid sequence occurs. 
     
     
         35 . A method of amplifying a nucleic acid sequence in a parallel sequencing technique, said method comprising:
 hybridizing a 3′ target specific region of a first linear primer to a first part of a target nucleic acid sequence, wherein the first linear primer comprises a universal priming region that is distinct from the 3′ target specific region, wherein the 3′ target specific region is a random region, wherein the first linear primer and the target nucleic acid sequence are within a volume of liquid that is more than 60 nl;   extending the first linear primer that is hybridized to the target nucleic acid sequence to form an extended linear primer;   hybridizing a second linear primer to a complementary part of the target nucleic acid sequence of the extended linear primer, wherein the second linear primer comprises a same universal priming region as the first linear primer;   extending the second linear primer to form a double-extended linear primer;   amplifying the double-extended linear primer using an amplification primer, wherein the amplification primer comprises a sequence that is the same as a sequence of the universal priming region, wherein a double-extended linear primer comprising a first insert section is selectively amplified over a double-extended linear primer comprising a second insert section;   adding a third primer that comprises a sequence that is complementary to a sequence within the first insert section;   performing an amplification reaction using the third primer to amplify the target nucleic acid sequence within the first insert section of the double-extended linear primer, wherein the double-extended linear primer comprising the first insert has its insert section selectively amplified compared to the double-extended linear primer comprising the second insert section, wherein the first insert section is larger than the second insert section; and   performing a parallel sequencing reaction on an amplified product from the amplification reaction using the third primer.   
     
     
         36 . A method of amplifying a nucleic acid sequence in a parallel sequencing technique, said method comprising:
 hybridizing a 3′ target specific region of a first linear primer to a first part of a target nucleic acid sequence, wherein the first linear primer comprises a universal priming region that is separate from the 3′ target specific region, wherein the 3′ target specific region is a random region, wherein the random region comprises at least one nucleotide that is not an adenine or a guanine;   extending the first linear primer that is hybridized to the target nucleic acid sequence to form an extended linear primer;   hybridizing a second linear primer to a complementary part of the target nucleic acid sequence of the extended linear primer, wherein the second linear primer comprises a same universal priming region as the first linear primer;   extending the second linear primer to form a double-extended linear primer;   amplifying the double-extended linear primer using an amplification primer, wherein the amplification primer comprises a sequence that is the same as a sequence of the universal priming region, wherein a double-extended linear primer comprising a first insert section is selectively amplified over a double-extended linear primer comprising a second insert section because the double-extended linear primer comprising a second insertion section forms a self-hybridized structure;   adding a third primer that comprises a sequence that is complementary to a sequence within the first insert section;   adding a fourth primer that comprises a sequence that is complementary to a sequence within the first insert section;   performing an amplification reaction using the third and fourth primers to amplify the target nucleic acid sequence within the first insert section of the double-extended linear primer, wherein the double-extended linear primer comprising the first insert has its insert section selectively amplified compared to the double-extended linear primer comprising the second insert, wherein the first insert section is larger than the second insert section; and   performing a parallel sequencing reaction on an amplified product from the amplification reaction using the third and fourth primers.   
     
     
         37 . A method of selectively amplifying a nucleic acid sequence comprising:
 hybridizing a 3′ target specific region of a first primer to a first part of a target nucleic acid sequence, wherein the first primer comprises a universal priming region that is distinct from the 3′ target specific region, wherein the 3′ target specific region is a random region;   extending the first primer that is hybridized to the target nucleic acid sequence to form an extended primer;   hybridizing a second primer to a complementary part of the target nucleic acid sequence of the extended primer, wherein the second primer comprises a same universal priming region as the first primer;   extending the second primer to form a double-extended primer;   amplifying the double-extended primer using an amplification primer, wherein the amplification primer comprises a sequence that is the same as a sequence of the universal priming region, wherein a double-extended primer comprising a first insert section that is at least 100 nucleotides in length is selectively amplified over a double-extended primer comprising a second insert section that is less than 100 nucleotides in length;   adding a third primer that comprises a sequence that is complementary to a sequence within the first insert section;   adding a fourth primer that comprises a sequence that is complementary to a sequence within the first insert section; and   performing an amplification reaction using the third primer and fourth primer to amplify the target nucleic acid sequence within the first insert section of the double-extended primer, wherein the double-extended primer comprising the first insert has its insert section selectively amplified compared to the double-extended primer comprising the second insert section.