US 5,851,805 AGranted

Method for producing DNA from mRNA

AssigneeBoard of Trustees operating Michigan State University

InventorsDong Wei, Veronica M. Maher, Joseph Justin McCormick

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Abstract

An improved method for amplifying cDNA fragments corresponding to the mRNA species expressed in cells is described. The method involves priming the mRNA with a first labeled synthetic oligodeoxynucleotide and then reverse transcribing the mRNA to produce a mRNA:cDNA hybrid; and then producing a double-stranded DNA from the hybrid. This DNA is digested with a restriction endonuclease and then a synthetic DNA linker, carrying a second label, is ligated at the cut to the DNA strand containing the 5' phosphate group. The DNA is then isolated with a first reagent which binds the second label. The DNA fragments of interest are then isolated using a second reagent which binds the first label. The isolated DNA fragments are amplified using polymerase chain reaction, and then separated on a sequencing gel. The method is particularly useful for comparing the mRNA species expressed in the cells of various samples.

Claims27

Dependent claims

A method for producing DNA from mRNA in a sample, which comprises:

(a) isolating RNA from the sample;
(b) priming the mRNA with a first synthetic oligodeoxynucleotide that hybridizes with the polyadenylate (polyA) tail of mRNA and one non-adenylate nucleotide, and consists of polydeoxythymidylate (dT) with a 5' and a 3' end, which has a first label at the 5' end and has a deoxynucleotide (M) at the 3' end, wherein M is selected from the group consisting of dG, dC and dA;
(c) reverse transcribing the mRNA to produce a mRNA:cDNA hybrid;
(d) producing a double-stranded DNA from the mRNA:cDNA hybrid;
(e) digesting the double-stranded DNA with a restriction endonuclease to produce a cut in the double-stranded DNA;
(f) ligating the 5' end of one strand of the double-stranded DNA at the cut to a 3' end of one strand of a complementary pair of second and third synthetic oligodeoxynucleotides that comprise a linker DNA, where one of the strands of the linker DNA is longer than the other, so that the linker DNA has a staggered end and a blunt end, wherein the longer strand of the linker DNA has a second label, different from the first label, at the staggered end, the 5' end, and is the strand of the linker DNA that is ligated at the blunt end to the polyA tail strand of the double-stranded DNA at the cut;
(g) isolating the double-stranded DNA containing the second label with a first reagent which binds the second label to retain the DNA that has been ligated to the linker DNA;
(h) isolating the double-stranded DNA containing the first label and second label with a second reagent which binds the first label, wherein the isolated DNA is now free of double stranded DNA containing only the second label;
(i) amplifying the isolated DNA strand in step (h) which contains the polyA tail by a polymerase chain reaction (PCR);
and
(j) separating the amplified DNA.

8 more elements

A method for identifying mRNA produced by a cell population which comprises:

(a) isolating RNA from a population of cells;
(b) priming the mRNA with a synthetic oligodeoxynucleotide that hybridizes to the polyA tail of the mRNA and has the formula (dT).sub.x M which has a first label at its 5' end, wherein dT is deoxythymidylate, M is selected from the group consisting of dG, dC and dA and x is a whole number between 20 and 28;
(c) reverse transcribing the mRNA to produce a mRNA:cDNA hybrid;
(d) producing a double-stranded DNA from the mRNA:cDNA hybrid;
(e) digesting the double-stranded DNA with a restriction endonuclease to produce a cut in the double-stranded DNA;
(f) ligating the 5' end of one strand of the double-stranded DNA at the cut to a 3' end of one strand of a complementary pair of second and third synthetic oligodeoxynucleotides that comprise a linker DNA, where one of the strands of the linker DNA is longer than the other, so that the linker DNA has a staggered end and a blunt end, wherein the longer strand of the linker DNA has a second label, different from the first label, at its staggered end, the 5' end, and is the strand of the linker DNA that is ligated at the blunt end to the polyA tail strand of the double-stranded DNA at the cut;
(g) isolating the double-stranded DNA containing the second label with a first reagent which binds the second label to retain the DNA that has been linked to the linker DNA;
(h) isolating the double-stranded DNA containing the first label and second label with a second reagent which binds the first label on double stranded DNA, wherein the isolated DNA is now free of double stranded DNA containing only the second label;
(i) amplifying the isolated DNA strand in step (h) which contain the polyA tail by a polymerase chain reaction (PCR);
and
(j) separating the amplified DNA, wherein the DNA identifies the mRNA produced by the population of cells.

8 more elements

A method for identifying mRNA produced by a set of cell populations which comprises:

(a) isolating RNA from one of the cell populations;
(b) priming the mRNA with a synthetic oligodeoxynucleotide that hybridizes to the polyA tail of the mRNA and has the formula (dT).sub.x M which has a first label at its 5' end, wherein dT is deoxythymidylate, M is selected from the group consisting of dG, dC and dA and x is a whole number between 20 and 28;
(c) reverse transcribing the mRNA to produce a mRNA:cDNA hybrid;
(d) producing a double-stranded DNA from the mRNA:cDNA hybrid;
(e) digesting the double-stranded DNA with a restriction endonuclease to produce a cut in the double-stranded DNA;
(f) ligating the 5' end of one strand of the double-stranded DNA at the cut to a 3' end of one strand of a complementary pair of second and third synthetic oligodeoxynucleotides that comprise a linker DNA, where one of the strands of the linker DNA is longer than the other, so that the linker DNA has a staggered end and a blunt end, wherein the longer strand of the linker DNA has a second label, different from the first label, at the staggered end, the 5' end, and is the strand of the linker DNA that is ligated at the blunt end to the polyA tail strand of the double-stranded DNA at the cut;
(g) isolating the double-stranded DNA containing the second label with a first reagent which binds the second label to retain the DNA that has been ligated to the linker DNA;
(h) isolating the double-stranded DNA containing the first label and second label with a second reagent which binds the first label, wherein the isolated DNA is now free of double stranded DNA containing only the second label;
(i) amplifying the isolated DNA strand in step (h) which contains the polyA tail by a polymerase chain reaction (PCR);
(j) separating the amplified DNA, wherein the DNA identifies the mRNA produced by the cell population;
and
(k) repeating steps (a) to (j) with different endonucleases to detect differences in the mRNA in the set of cell populations.

9 more elements

A method for producing DNA from mRNA in a sample, which comprises:

(a) isolating RNA from the sample;
(b) priming the mRNA with a first synthetic oligodeoxynucleotide that hybridizes with the polyadenylate (polyA) tail of mRNA and one non-polyadenylate nucleotide and consists of polydeoxythymidylate (polydT) with a 5' and a 3' end, which has a first label at the 5' end and has a deoxynucleotide (M) at the 3' end, wherein M is selected from the group consisting of dG, dC and dA;
(c) reverse transcribing the mRNA to produce an mRNA:cDNA hybrid;
(d) producing a double-stranded DNA from the mRNA:cDNA hybrid;
(e) digesting the double-stranded DNA with a restriction endonuclease to produce a cut in the double-stranded cDNA;
(f) ligating the 5' end of one strand of the double-stranded DNA at the cut to a 3' end of one strand of a complementary pair of second and third synthetic oligodeoxynucleotides that comprise a linker DNA, where one of the strands of the linker DNA is longer than the other, so that the linker DNA has a staggered end and a blunt end, wherein the longer strand of the linker DNA has a second label, different from the first label, at the staggered end, the 5' end, and is the strand of the linker DNA that is ligated at the blunt end to the strand of the double-stranded DNA at the cut;
(g) isolating the cDNA strand containing the second label free of the cDNA strand containing the first label, comprising the steps of:
(1) isolating the double-stranded DNA with a first reagent which binds the second label to remove the double-stranded cDNA that has not been cut by the restriction enzyme and therefore has not been ligated to the linker DNA
(2) isolating the double-stranded DNA containing the polyA tail with a second reagent which binds the first label to eliminate double-stranded cDNA that contains only the second label, then
(3) isolating the DNA strand containing the second label free of the cDNA strand containing the first label;
(h) amplifying the isolated DNA strand containing the polyA tail by a polymerase chain reaction (PCR);
and
(i) separating the amplified DNA.

10 more elements

Dependencies are inferred from the claim text.