Selective terminal tagging of nucleic acids

Abstract

Methods are provided for adding a terminal sequence tag to nucleic acid molecules for use in RNA or DNA amplification. The tag introduced may be used as a primer binding site for subsequent amplification of the DNA molecule and/or sequencing of the DNA molecule and therefore provides means for identification and cloning of the 5′-end or the complete sequence of mRNAs.

1 . A method for adding a terminal sequence tag to a target DNA molecule, the method comprising:
 A) providing:
 1) a sample containing at least one target DNA molecule; 
 2) a first oligonucleotide comprising deoxyribonucleotides, wherein:
 (a) the 5′ overhanging portion exhibits a user-defined first sequence tag; 
 (b) the 3′ hybridizing portion that is capable of hybridizing to the 3′-end of each of the at least one target DNA molecule; and 
 (c) the 3′-end is blocked; and 
 
 3) a first DNA polymerase; 
   B) contacting the at least one target DNA molecule with the first oligonucleotide under conditions allowing hybridization of the 3′ hybridizing portion of the first oligonucleotide to the at least one target DNA molecule; and   C) incubating the at least one target DNA molecule to which the first oligonucleotide is hybridized with the DNA polymerase under conditions wherein the 3′ end of the at least one target DNA molecule is extended using the 5′ overhanging portion of the first oligonucleotide as a template to generate a first DNA template that exhibits a terminal sequence tag that is complementary to the first sequence tag of the first oligonucleotide.   
     
     
         2 . The method of  claim 1 , wherein the sample that contains the at least one target DNA molecule provided in step A)1) contains a plurality of target DNA molecules and the first oligonucleotide provided in step A)2) comprises a plurality of first oligonucleotides wherein the 3′ hybridizing portion of at least one of the first oligonucleotides in the plurality of first oligonucleotides exhibits a sequence that is capable of hybridizing to the 3′ end of each of the target DNA molecules in the plurality of target DNA molecules. 
     
     
         3 . The method of  claim 2 , wherein the 3′ hybridizing portions of the plurality of first oligonucleotides exhibit random sequences that, together, comprise all possible sequences for the 3′ hybridizing portion. 
     
     
         4 . The method of  claim 1 , wherein the at least one target DNA molecule is complementary DNA (cDNA) generated by reverse transcription of at least one RNA molecule. 
     
     
         5 . The method of  claim 4 , wherein the at least one target DNA molecule comprises cDNA generated by reverse transcription of messenger RNA. 
     
     
         6 . The method of  claim 5 , wherein the reverse transcription of messenger RNA is performed using a reverse transcription primer selected from among an oligo(dT) primer and an oligo(dT)nV primer. 
     
     
         7 . The method of  claim 1 , wherein at least one target DNA molecule is of unknown sequence. 
     
     
         8 . The method of  claim 1 , wherein at least one target DNA molecule is of known sequence. 
     
     
         9 . The method of  claim 1 , further comprising step D) of: separating or removing unused or unhybridized first oligonucleotide and first oligonucleotide that is hybridized to the first DNA template. 
     
     
         10 . The method of  claim 9 , wherein the separating or removing of the first oligonucleotide in step D) comprises using a size selection column and buffers. 
     
     
         11 . The method of  claim 1 , wherein the first sequence tag of the first oligonucleotide does not exhibit a sequence of an RNA polymerase promoter, wherein the method further comprises:
 A) providing:
 4) a second oligonucleotide comprising, sequentially in a 5′ to 3′ direction:
 (a) a 5′-overhanging portion that comprises a second sequence tag that exhibits a sequence of one strand of an RNA polymerase promoter; and 
 (b) a 3′ hybridizing portion that exhibits a sequence that is identical to a sequence exhibited by the first sequence tag of the 5′ overhanging portion of the first oligonucleotide; 
 
 5) a second DNA polymerase; and 
 6) an RNA polymerase that is capable of binding to an RNA polymerase promoter comprising the RNA polymerase promoter sequence exhibited by the 5′-overhanging portion of the second oligonucleotide; 
   D) separating or removing excess unhybridized first oligonucleotide and oligonucleotide that is hybridized to the first DNA template;   E) contacting the first DNA template with the second oligonucleotide under conditions allowing hybridization or annealing of the 3′ hybridizing portion of the second oligonucleotide to the 3′ end of the first DNA template that has the terminal sequence tag that is complementary to the first sequence tag of the first oligonucleotide;   F) incubating the first DNA template to which the second oligonucleotide is hybridized with the DNA polymerase under conditions wherein the 3′ end of the first DNA template is extended using the 5′ overhanging portion of the second oligonucleotide as a template and the 3′ end of the second oligonucleotide is extended using the first DNA template as a template to generate a new second DNA template, thereby generating double-stranded DNA that contains a double-stranded RNA polymerase promoter;   G) contacting the double-stranded DNA with the RNA polymerase and incubating under in vitro transcription conditions wherein RNA is synthesized.   
     
     
         12 . The method of  claim 11 , wherein the second DNA polymerase is the same as the first DNA polymerase. 
     
     
         13 . The method of  claim 11 , wherein the separating or removing of the first oligonucleotide in step D) comprises using a size selection column and buffers. 
     
     
         14 . The method of  claim 11 , wherein the double-stranded DNA comprising the first DNA template and the second DNA template generated in step F) is purified using a size selection column. 
     
     
         15 . The method of  claim 11 , wherein the method further comprises purifying the RNA by: contacting the RNA synthesized in step G) with DNase under conditions wherein DNA is digested; phenol extraction; or using a size selection column. 
     
     
         16 . The method of  claim 11 , wherein the method further comprises making cDNA from the RNA synthesized in step G) to produce a second round of first DNA templates. 
     
     
         17 . The method of  claim 16 , wherein the method further comprises: contacting the second-round first DNA templates with the second oligonucleotides and the first or second DNA polymerase under conditions wherein the 3′ terminal sequence tags of the second-round first DNA templates hybridize to the 3′ portions of the second oligonucleotides, whereupon, the 3′ end of the second-round first DNA templates and the hybridized second oligonucleotides are each extended by the DNA polymerase to generate a second round of double-stranded DNA that contains a double-stranded RNA polymerase promoter and that comprises second-round first and second DNA templates. 
     
     
         18 . The method of  claim 17 , wherein the method further comprises: contacting the second-round double-stranded DNA with the RNA polymerase under conditions wherein a second round of RNA is synthesized, each molecule of which exhibits a sense-strand sequence of an RNA molecule complementary to a target DNA in the sample. 
     
     
         19 . The method of  claim 11 , wherein step F) further comprises:
 PCR amplifying the amount of double-stranded DNA which encodes at least one RNA of interest in the sample by incubating the double-stranded DNA generated in step F) under PCR amplification conditions with a thermostable DNA polymerase in the presence of a first PCR primer that exhibits a sequence that is identical to the 5′ end portion of the second DNA template and a second PCR primer that exhibits a sequence that is complementary to a 3′ terminal poly(A) tail or coding sequence of each RNA molecule of interest in the sample.   
     
     
         20 . A method for tagging the 3′ end of at least one DNA molecule having a 3′ end portion of unknown sequence, comprising:
 A) providing:
 1) a sample comprising at least one DNA molecule having a 3′ end portion of unknown sequence; 
 2) a plurality of first oligodeoxyribonucleotides, each molecule of which comprises:
 (a) a 5′ overhanging portion that exhibits a user-defined first sequence tag, 
 (b) a 3′ end portion that exhibits a random sequence, and 
 (c) a 3′ end that is blocked so as not to be extendible by a DNA polymerase; 
 
 3) a DNA polymerase; 
 
 B) combining the sample, the plurality of first oligodeoxyribonucleotides, and the DNA polymerase in a mixture under conditions in which:
 1) the 3′ end portion of the at least one DNA molecule hybridizes to a sequence in the 3′ end portion of a first oligodeoxyribonucleotide, 
 2) the 3′ end portion of the at least one DNA molecule is extended by the DNA polymerase using the 5′ overhanging portion of the first oligonucleotide as a template, thereby tagging the 3′ end of the at least one DNA molecule in the sample with a second sequence tag that is complementary to the first sequence tag in the 5′ overhanging portion of the first oligonucleotide, and 
 3) the first oligodeoxyribonucleotide is not extended by the DNA polymerase.