Methods and means for manipulating nucleic acid
First Claim
1. A method of providing a population of double-stranded product DNA molecules, the method comprising:
- annealing polyA tails of mRNA molecules in a sample to an oligoT adaptor, which oligoT adaptor comprises a 3′
oligoT portion and a 5′
first back primer annealing sequence, synthesizing a cDNA. strand complementary to the mRNA molecules using the mRNA molecules as template, thereby providing a population of first cDNA strands;
removing the mRNA;
synthesizing a second cDNA strand complementary to each first strand, thereby providing a population of double-stranded cDNA molecules;
digesting the double-stranded cDNA molecules with a Type II or Type IIS restriction enzyme to provide a population of digested double-stranded cDNA molecules, each digested double-stranded cDNA molecule having a cohesive end provided by the restriction enzyme digestion;
ligating a population of cohesive adaptor oligonucleotides to the cohesive end of each of the digested double-stranded cDNA molecules, the cohesive adaptor oligonucleotides each comprising an end sequence complementary to a cohesive end, a first forward primer annealing sequence, and a second forward primer annealing sequence between the first forward primer annealing sequence and the cohesive end, thereby providing double-stranded template cDNA molecules each comprising a first strand and a second strand wherein the first strand of the double-stranded template cDNA molecules each comprise a 3′
terminal cohesive adaptor oligonucleotide and the second strand of the double-stranded template cDNA molecules each comprise a 3′
sequence complementary to the oligot adaptor sequence;
purifying said double-stranded template cDNA molecules;
performing a first polymerase chain reaction on the double-stranded template cDNA molecules having a sequence complementary to a 3′
end of an mRNA using a first forward primer, which comprises a sequence which anneals to the first forward primer annealing sequence, and a first back primer, which comprises a sequence which anneals to the first back primer annealing sequence;
performing a second polymerase chain reaction amplification on products of the first polymerase chain reaction using a population of second forward primers and a population of second back primers, wherein the second forward primers each comprise a sequence which anneals to a second forward primer annealing sequence of a cohesive adaptor oligonucleotide; and
where the restriction enzyme is a Type II enzyme the second forward primers each comprise at least one 3′
terminal variable nucleotide and optionally more than one 3′
terminal variable nucleotides wherein the variable nucleotide is, or at a corresponding position within the variable nucleotides each second forward primer has, a nucleotide selected from A, T, C and G, whereby the population of second forward primers primes synthesis in the polymerase chain reaction of first strand product DNA molecules each of which is complementary to the first strand of a template cDNA molecule that comprises adjacent to the primer annealing sequence within the first strand of the template CDNA molecule a nucleotide or sequence of nucleotides complementary to the variable nucleotide or nucleotides of a second forward primer within the population of second forward primers;
or where the restriction enzyme is a Type IIS enzyme the second forward primers prime synthesis in the polymerase chain reaction of first strand product DNA molecules each of which is complementary to the first strand of a template cDNA molecule that comprises within the first strand of the template cDNA molecule a sequence of nucleotides complementary to an end sequence of a cohesive adaptor oligonucleotide in the population of cohesive adaptor oligonucleotides;
the second back primers comprise an oligoT sequence and a 3′
variable portion conforming to the following formula;
(G/C/A) (X)n wherein X is any nucleotide, n is zero, at least one or more than one;
whereby the population of second back primers primes synthesis in the polymerase chain reaction of second strand product DNA molecules each of which is complementary to the second strand of a template cDNA molecule that comprises adjacent to polyA within the second strand of the template cDNA molecule a nucleotide or nucleotides complementary to the variable portion of a second back primer within the population of second back primers;
whereby performing the polymerase chain reaction amplifications provides a population of double-stranded product DNA molecules each of which comprises a-first strand product DNA molecule and a second strand product DNA molecule.
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Abstract
Methods of manipulation of nucleic acid, in particular amplification by means of the polymerase chain reaction (PCR), including use of oligonucleotides and combinations and kits comprising such oligonucleotides, also methods comprising use of nested PCR, allowing for improved results in methods wherein large numbers of nucleic acid fragments are manipulated by means of PCR and electrophoresis. Oligonucleotides are provided for use a size standards in electrophoresis, and internal controls allowing for calculation of relative amounts of material present. Improved results can be achieved in methods of profiling mRNA transcribed in a system under investigation.
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Citations
18 Claims
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1. A method of providing a population of double-stranded product DNA molecules, the method comprising:
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annealing polyA tails of mRNA molecules in a sample to an oligoT adaptor, which oligoT adaptor comprises a 3′
oligoT portion and a 5′
first back primer annealing sequence, synthesizing a cDNA. strand complementary to the mRNA molecules using the mRNA molecules as template, thereby providing a population of first cDNA strands;
removing the mRNA;
synthesizing a second cDNA strand complementary to each first strand, thereby providing a population of double-stranded cDNA molecules;
digesting the double-stranded cDNA molecules with a Type II or Type IIS restriction enzyme to provide a population of digested double-stranded cDNA molecules, each digested double-stranded cDNA molecule having a cohesive end provided by the restriction enzyme digestion;
ligating a population of cohesive adaptor oligonucleotides to the cohesive end of each of the digested double-stranded cDNA molecules, the cohesive adaptor oligonucleotides each comprising an end sequence complementary to a cohesive end, a first forward primer annealing sequence, and a second forward primer annealing sequence between the first forward primer annealing sequence and the cohesive end, thereby providing double-stranded template cDNA molecules each comprising a first strand and a second strand wherein the first strand of the double-stranded template cDNA molecules each comprise a 3′
terminal cohesive adaptor oligonucleotide and the second strand of the double-stranded template cDNA molecules each comprise a 3′
sequence complementary to the oligot adaptor sequence;
purifying said double-stranded template cDNA molecules;
performing a first polymerase chain reaction on the double-stranded template cDNA molecules having a sequence complementary to a 3′
end of an mRNA using a first forward primer, which comprises a sequence which anneals to the first forward primer annealing sequence, and a first back primer, which comprises a sequence which anneals to the first back primer annealing sequence;
performing a second polymerase chain reaction amplification on products of the first polymerase chain reaction using a population of second forward primers and a population of second back primers, wherein the second forward primers each comprise a sequence which anneals to a second forward primer annealing sequence of a cohesive adaptor oligonucleotide; and
where the restriction enzyme is a Type II enzyme the second forward primers each comprise at least one 3′
terminal variable nucleotide and optionally more than one 3′
terminal variable nucleotides wherein the variable nucleotide is, or at a corresponding position within the variable nucleotides each second forward primer has, a nucleotide selected from A, T, C and G, whereby the population of second forward primers primes synthesis in the polymerase chain reaction of first strand product DNA molecules each of which is complementary to the first strand of a template cDNA molecule that comprises adjacent to the primer annealing sequence within the first strand of the template CDNA molecule a nucleotide or sequence of nucleotides complementary to the variable nucleotide or nucleotides of a second forward primer within the population of second forward primers;
orwhere the restriction enzyme is a Type IIS enzyme the second forward primers prime synthesis in the polymerase chain reaction of first strand product DNA molecules each of which is complementary to the first strand of a template cDNA molecule that comprises within the first strand of the template cDNA molecule a sequence of nucleotides complementary to an end sequence of a cohesive adaptor oligonucleotide in the population of cohesive adaptor oligonucleotides;
the second back primers comprise an oligoT sequence and a 3′
variable portion conforming to the following formula;
(G/C/A) (X)n wherein X is any nucleotide, n is zero, at least one or more than one;
whereby the population of second back primers primes synthesis in the polymerase chain reaction of second strand product DNA molecules each of which is complementary to the second strand of a template cDNA molecule that comprises adjacent to polyA within the second strand of the template cDNA molecule a nucleotide or nucleotides complementary to the variable portion of a second back primer within the population of second back primers;
whereby performing the polymerase chain reaction amplifications provides a population of double-stranded product DNA molecules each of which comprises a-first strand product DNA molecule and a second strand product DNA molecule. - View Dependent Claims (2, 3, 4, 5, 6, 8, 9, 10, 11)
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7. A method of amplifying cDNA fragments to provide a population of double-stranded product DNA molecules, each cDNA fragment comprising an upper strand that comprises a copy of a 3′
- fragment of an mRNA molecule comprising a polyA tail, and a lower strand that is complementary to the upper strand, wherein the upper strand comprises at its 5′
terminus the following adaptor (1) sequence;
5′
-AGGACATTTGTGAGTCAGGCGTGTCTTGGATGC-3′
, and the lower strand comprises at its 3′
terminus the following adaptor (2) sequence;
5′
-p(N)xGCATCCAAGACACGCCTGACTCACAAATGTCCT-3′
, and wherein the lower strand comprises at its 5′
terminus the following adaptor (3) sequence;
5′
-CCAATTCACGCTGGACTGTTTCGG-(T)y-3′ and
the upper strand comprises at its 3′
terminus the following adaptor (4) sequence;
5′
-(A)y-CCGAAACAGTCCAGCGTGAATTGG-3′
,wherein the upper and lower strands.are provided by ligation of adaptors of adaptor sequence (1) and (2) following restriction digest of cDNA fragments, wherein N is A, T, C or G, and wherein x corresponds to the number of bases of overhang created by the restriction digest;
the method comprising performing nested polymerase chain reaction, wherein a first polymerase chain reaction is performed with a first forward primer of the following sequence;
5′
-AGGACATTTGTGAGTCAGGC-3′
(SEQ ID NO.
26), and a first back primer of the following sequence;
5′
-TTCACGCTGGACTGTTTCGG-3′
(SEQ ID NO.
27), andwherein a second polymerase chain reaction is performed with a second forward primer of the following sequence;
5′
-GTGTCTTGGATGC-3′
(SEQ ID NO.
35), and a second back primer of the following sequence;
5′
-(T)zVN1N2, wherein z is 10-40, V is A, G or C, N1 is optional and if present is A, G, C or T, and N2 is optional and if present is A, G, C or T.
- fragment of an mRNA molecule comprising a polyA tail, and a lower strand that is complementary to the upper strand, wherein the upper strand comprises at its 5′
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12. A set of primers for nested polymerase chain reaction to amplify cDNA copies of mRNA fragments comprising polyA tails, wherein the set comprises
a first forward primer of the following sequence: - 5′
-AGGACATTTGTGAGTCAGGC-3′
(SEQ ID NO.
26),a first back primer of the following sequence;
5′
-TTCACGCTGGACTGTTTCGG-3′
(SEQ ID NO.
27),a second forward primer of the following sequence;
5′
-GTGTCTTGGATGC-3′
(SEQ ID NO.
35), anda second back primer of the following sequence;
5′
-(T)zVN1N2, wherein z is 10 to 40, V is A, G or C,N1 is optional and if present is A, G, C or T, and N2 is optional and if present is A, G, C or T. - View Dependent Claims (13, 14, 15, 16, 17, 18)
- 5′
Specification