Process for producing cDNA

• US 5,759,820 A
• Filed: 07/25/1994
• Issued: 06/02/1998
• Est. Priority Date: 11/21/1988
• Status: Expired due to Term

• Current Assignee: Life Technologies AS (Perspecta, Inc.)
• Original Assignee: Dynel Comapny
• Inventors: Hornes, Erik, Korsnes, Lars
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/280,133
• Time in Patent Office: 1,408 Days
• Field of Search: 435/6, 435/91.2, 435/5, 435/91.1, 435/173.9, 436/125, 436/126, 204/660, 204/156, 204/554, 422/186.01, 422/186.03, 536/24.3-24.33
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: Y10T 436/193333 In aqueous solution
• CPC Class Codes: Y10T 436/196666 Carbon containing compound [e.g., vinylchloride, etc.]

Abstract

The process includes the steps of: a) contacting a liquid containing mRNA with an insoluble support having DNA probes attached thereto via the 5'"'"' -terminus thereof whereby the MRNA is hybridized to said probes and hence to said support; b) removing said liquid; and c) adding enzymes and nucleotides in solution whereby the probe functions as a primer to produce single stranded cDNA on the mRNA probe. The probe DNA may be oligo-dT or a specific DNA sequence, such as one that is complementary to a conserved region in a class of mRNA molecules. The insoluble support comprises magnetic particles which are monodisperse polymer particles comprising superparamagnetic iron oxide, a coating to reduce non-specific binding and a substituent for attaching an oligonucleotide.

Claims

1. A process for the production of cDNA, comprising the steps of:

(a) contacting a liquid containing mRNA with an insoluble support having DNA probes attached thereto via their 5'"'"' termini whereby mRNA in said sample is hybridized to said probes and thereby bound to said support, wherein;

(A) said insoluble support comprises a plurality of particles that are comprised of;

(i) superparamagnetic iron oxide dispersed within a polymer particle, (ii) nucleic acid-binding functional groups at the surface of said particle for bonding said probes and (iii) a coating to reduce non-specific binding;

(B) said probes are bound to said particles via said functional group;

(C) the particles in said plurality are monodisperse and the standard deviation of the diameter is less than 5%;

(b) removing said liquid; and

(c) adding enzymes and nucleotides in solution whereby said probe functions as a primer to produce single stranded cDNA on the mRNA templates.

2. A process as claimed in claim 1 in which the DNA probes comprise at their 5'"'"' ends a DNA sequence containing at least one restriction site.

3. A process as claimed in claim 1 wherein the probe is oligo-dT.

4. A process as claimed in claim 1 wherein the probe binds in a sequence specific manner to a target mRNA sequence.

5. A process as claimed in claim 4, in which the probe comprises a specific DNA sequence which is complementary to a sequence which occurs in a multiplicity of different mRNAs.

6. A process as claimed in claim 1 wherein the single stranded cDNA is subsequently converted into double stranded DNA.

7. A process as claimed in claim 1 for producing a cDNA subtraction library from two populations of cells which express mRNAs which differ in sequence, comprising the steps of:

a) contacting a liquid containing the total mRNA from a first one of said cell populations with said insoluble support carrying 5'"'"'-attached oligo-dT probes whereby said mRNA is hybridized thereto;

b) removing said liquid;

c) using said oligo-dT probes as a primer and adding enzymes and nucleotides in solution whereby corresponding single strands of cDNA are formed and removing the hybridized mRNA to provide a first strand cDNA library attached to said insoluble support;

d) contacting the insoluble support carrying said attached first strand cDNA with a liquid containing the total mRNA of the second one of said two cell populations to hybridize mRNA present in both cell populations and

e) removing the insoluble support and attached mRNA to leave in solution those mRNA molecules present only in said second cell population that do not hybridize to said cDNA;

f) making said cDNA subtraction library from said mRNA remaining in solution.

8. A process as claimed in claim 7 wherein the insoluble support bearing the cDNA library at step c) is treated with oligo-dA before step d) to avoid non-specific mRNA binding.

9. A process as claimed in claim 1 which includes washing steps and changes of buffer to optimise hybridisation and enzyme activity.

10. A kit for cDNA synthesis comprising:

(a) an insoluble support having DNA probes attached thereto, and one or more of the following;

(b) a reverse transcriptase;

(c) a polymerase and optionally a ligase and/or a nuclease;

(d) deoxynucleotides, and

(e) buffers suitable for reverse transcription and/or polymerization

wherein said insoluble support comprises a plurality of particles that are comprised of;

(i) superparamagnetic iron oxide dispersed within a polymer particle, (ii) a nucleic acid-binding functional group at the surface of said particle for bonding said probes and (iii) a coating to reduce non-specific binding;

said probes are bound to said particles via said functional group, and the particles in said plurality are monodisperse and the standard deviation of their diameter is less than 5%.

11. A process for the production of cDNA, comprising the steps of:

(a) contacting a liquid containing mRNA with monodisperse, superparamagnetic particles carrying a single stranded 5'"'"'-attached DNA probe capable of binding to said mRNA;

(b) removing said liquid;

(c) contacting said particles with an aqueous solution of a reverse transcriptase or polymerase in the presence of labelled nucleotides using the probe as a primer whereby labelled complementary single stranded DNA is formed if said mRNA was present in said liquid, wherein a predetermined amount of dideoxyunucleotides are present to limit chain synthesis to a predetermined average length thereby enabling the total amount of label to be correlated with the number of target nucleic acid molecules;

(d) magnetically aggregating said particles onto a surface and removing said solution; and

(e) determining the amount of said label in said complementary single stranded DNA.

12. A process as claimed in claim 11 for producing a cDNA subtraction library from two populations of cells which differ in their mRNAs, comprising the steps of:

(a) contacting a liquid containing total mRNA from a first one of said cell populations with said monodisperse superparamagnetic particles carrying 5'"'"'-attached oligo-dT probes whereby substantially all said m A is hybridized thereto;

(b) removing said liquid;

(c) using said oligo-dT probes as a primer and adding enzymes and nucleotides in solution whereby corresponding single strands of cDNA are formed and removing the hybridized mRNA to provide a first strand cDNA library attached to said monodisperse superparamagnetic particles;

(d) contacting the monodisperse superparamagnetic particles carrying said attached first strand cDNA with a liquid containing total mRNA of a second one of said two cell populations to hybridize mRNA to the first strand cDNA; and

(e) removing the monodisperse superparamagnetic particles and attached mRNA to leave in solution those mRNA molecules present only in said second cell population that do not hybridize to said cDNA;

(f) making said cDNA subtraction library from said mRNA remaining in solution.