Molecular indexing for expressed gene analysis

• US 5,707,807 A
• Filed: 03/26/1996
• Issued: 01/13/1998
• Est. Priority Date: 03/28/1995
• Status: Expired due to Fees

• Current Assignee: Research Development Corporation Of Japan (Japan Science And Technology Agency)
• Original Assignee: Research Development Corporation Of Japan (Japan Science And Technology Agency)
• Inventors: Kato, Kikuya
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/621,914
• Time in Patent Office: 658 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/91.5, 435/91.2, 536/24.3, 536/24.33
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2537/149 Sequential reactions

Abstract

This invention relates to a method for classifying (indexing) cDNA which has been reverse-transcribed from tissue- or cell-derived RNA, or DNA in a short period without duplication by using class-IIS restriction enzymes or a combination of a class-IIS and a class-II restriction enzymes. According to this invention, it is possible to analyse and diagnose variations such as tumors easily, correctly and promptly by comparing the analyzed pattern of genes expressed in a cell or tissue sample with the analyzed pattern of normal genes. This method is also applicable to the search and isolation of genes of physiologically active substances that are potential pharmaceuticals or causative genes of hereditary diseases, as well as the isolation of those genes that are useful for improving agricultural products.

Claims

1. A method for determining the molecular index of a cDNA preparation comprising the following steps:

(a) digesting the cDNA preparation which has been reverse-transcribed by oligo-dT priming from a RNA preparation with a first class-IIS restriction enzyme;

(b) ligating each of the resultant cDNA fragments to an adaptor from a pool of 64 adapters cohesive to all possible overhangs of DNA fragments produced by the digestion in step (a) and thereby producing a ligation product, wherein each adaptor from said pool of adaptors has an overhang sequence and comprises a biotinylated oligonucleotide strand and a non-biotinylated oligonucleotide strand which has the overhang sequence;

(c) digesting the resultant ligation product with a second and a third class-IIS restriction enzymes which are different from the first class-IIS restriction enzyme used in step (a) and thereby obtaining a cDNA sample;

(d) recovering cDNA fragments which are ligated to adaptors from the cDNA samples by binding to streptavidin, removing non-biotinylated strands of polynucleotides from the recovered cDNA fragments, and thereby obtaining a single-stranded cDNA sample;

(e) amplifying the single-stranded cDNA sample by polymerase chain reaction using a first primer comprising a sequence identical to a segment or the whole of the biotinylated oligonucleotide strand of the adaptor and a second primer which is an anchor oligo-dT primer selected from the group consisting of d(T)₂₅ A, d(T)₂₅ C and d(T)₂₅ G, and thereby obtaining an amplification product; and

(f) separating the amplification product by denaturing polyacrylamide gel electrophoresis, recording the molecular sizes of DNA molecules in the amplification product, and thereby obtaining the molecular index of the cDNA preparation.

3. The method according to claim 1 or 2, wherein the class-IIS restriction enzymes are used in a combination of Fok I, Bsm AI and Bsm FI.

4. The method according to claim 1 or 2, wherein the cDNA preparation has been synthesized by reverse-transcribing RNA using as primer a mixture of the following oligonucleotides:

##STR6##

5. The method according to claim 4, wherein the amplification of the single-stranded cDNA sample is carried out using as the second primer an oligonucleotide selected from the group consisting of:

##STR7## instead of an anchored oligo-dT primer.

6. The method according to claim 1 or 2, wherein the first or second primer is labeled with a marker.

7. The method according to claim 5, wherein the marker is an radioactive atom, fluorescent dye or enzyme.

8. The method according to claim 1 or 2, wherein the streptavidin is attached to paramagnetic beads.

9. The method according to claim 1 or 2, wherein the RNA preparation is isolated from an animal cell or tissue, or a plant cell or tissue.

2. A method for determining the molecular index of a cDNA preparation comprising the following steps:

(a) digesting the cDNA preparation which has been reverse-transcribed by oligo-dT priming from a RNA preparation with a first class-IIS restriction enzyme;

(b) ligating each of the resultant cDNA fragments to an adaptor from a pool of 64 adapters cohesive to all possible overhangs of DNA fragments produced by the digestion in step (a) and thereby producing a ligation product, wherein each adaptor from said pool of adaptors has an overhang sequence and comprises a biotinylated oligonucleotide strand and a non-biotinylated oligonucleotide strand which has the overhang sequence;

(c) digesting the resultant ligation product with a second and a third class-IIS restriction enzymes which are different from the first class-IIS restriction enzyme used in step (a) and thereby obtaining a first cDNA sample;

(d) obtaining a second cDNA sample by repeating steps (a) to (c), wherein in step (a), the cDNA preparation is digested by the second class-IIS restriction enzyme, and in step (c), the ligation product is digested with the first and third class-IIS restriction enzymes;

(e) obtaining a third cDNA sample by repeating steps (a) to (c), wherein in step (a), the cDNA preparation is digested by the third class-IIS restriction enzyme, and in step (c), the ligation product is digested with the first and second class-IIS restriction enzymes;

(f) recovering cDNA fragments which are ligated to adaptors from the first, second and third cDNA samples by binding to streptavidin, removing non-biotinylated strands of polynucleotides from the recovered cDNA fragments, and thereby obtaining a single-stranded cDNA sample;

(g) amplifying the single-stranded cDNA sample by polymerase chain reaction using a first primer comprising a sequence identical to a segment or the whole of the biotinylated oligonucleotide strand of the adaptor and a second primer which is an anchor oligo-dT primer selected from the group consisting of d(T)₂₅ A, d(T)₂₅ C and d(T)₂₅ G, and thereby obtaining an amplification product; and

(h) separating the amplification product by denaturing polyacrylamide gel electrophoresis, recording the molecular sizes of DNA molecules in the amplification product, and thereby obtaining the molecular index of the cDNA preparation.

10. A method for determining the molecular index of a cDNA or DNA preparation comprising the following steps:

(a) digesting the cDNA or DNA preparation with a class-II restriction enzyme and thereby producing a first cDNA fragment preparation or first DNA fragment preparation, wherein the cDNA preparation has been reverse-transcribed by oligo-dT priming from a RNA preparation;

(b) ligating each cDNA fragment in the first cDNA preparation or each DNA fragment in the first DNA fragment preparation to a first adaptor which is cohesive to the overhang of cDNA fragments or DNA fragments produced by the class-II restriction enzyme and thereby producing a first ligated cDNA preparation or first ligated DNA preparation;

(c) digesting the first ligated cDNA preparation or first ligated DNA preparation with a class-IIS restriction enzyme and thereby producing a second cDNA fragment preparation or second DNA fragment preparation;

(d) ligating each cDNA fragment in the second cDNA fragment preparation or each DNA fragment in the second DNA fragment preparation to a second adaptor from a pool of 64 adapters cohesive to all possible overhangs of cDNA fragments or DNA fragments produced by the digestion with the class-IIS restriction enzyme and thereby producing a second ligated cDNA preparation or second ligated DNA preparation, wherein each second adaptor from said pool of adaptors has an overhang sequence and comprises a biotinylated oligonucleotide strand and a non-biotinylated oligonucleotide strand which has the overhang sequence;

(e) recovering cDNA fragments or DNA fragments which are ligated to adaptors from the second ligated cDNA preparation or second ligated DNA preparation by binding to streptavidin, removing non-biotinylated strands of polynucleotides from the recovered cDNA fragments or DNA fragments, and thereby obtaining a single-stranded cDNA or DNA sample;

(f) amplifying the single-stranded cDNA or DNA sample by polymerase chain reaction using a first primer comprising a sequence identical to a segment or the whole of the biotinylated oligonucleotide strand of the second adaptor and a second primer comprising a sequence identical or complementary to a segment or the whole of an oligonucleotide strand of the first adaptor, and thereby producing an amplification product; and

(g) separating the amplification product by denaturing polyacrylamide gel electrophoresis, recording the molecular sizes of amplified cDNA or DNA molecules in the amplification product, and thereby obtaining the molecular index of the cDNA or DNA preparation.

11. The method according to claim 10, wherein the class-IIS restriction enzyme is Fok I, Bsm AI, Bsm FI, Sfa NI or Bbv I.

12. The method according to claim 10, wherein the first or second primer is labeled with a marker.

13. The method according to claim 12, wherein the marker is an radioactive atom, fluorescent dye or enzyme.

14. The method according to claim 10, wherein the streptavidin is attached to paramagnetic beads.

15. The method according to claim 10, wherein the DNA preparation or RNA preparation is isolated from an animal cell or tissue, or a plant cell or tissue.