Method of differential display of exposed mRNA by RT/PCR

• US 5,599,672 A
• Filed: 12/08/1994
• Issued: 02/04/1997
• Est. Priority Date: 03/11/1992
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Dana-Farber Cancer Institute, President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Liang, Peng, Bianchi, Cesario F., Pardee, Arthur B.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/351,748
• Time in Patent Office: 789 Days
• Field of Search: 435/6, 435/91.2, 536/24.33, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A method for isolating mRNAs as cDNAs employs a polymerase amplification method using at least two oligodeoxynucleotide primers. In one approach, the first primer contains sequence capable of hybridizing to a site immediately upstream of the first A ribonucleotide of the mRNA'"'"'s polyA tail and the second primer contains arbitrary sequence. In another approach, the first primer contains sequence capable of hybridizing to a site including the mRNA'"'"'s polyA signal sequence and the second primer contains arbitrary sequence. In another approach, the first primer contains arbitrary sequence and the second primer contains sequence capable of hybridizing to a site including the Kozak sequence. In another approach, the first primer contains a sequence that is substantially complementary to the sequence of a mRNA having a known sequence and the second primer contains arbitrary sequence. In another approach, the first primer contains arbitrary sequence and the second primer contains sequence that is substantially identical to the sequence of a mRNA having a known sequence. The first primer is used as a primer for reverse transcription of the mRNA and the resultant cDNA is amplified with a polymerase using both the first and second primers as a primer set.

Claims

1. A method of comparing the presence or level of individual mRNA molecules in two or more nucleic acid samples, comprising the steps of:

providing a first nucleic acid sample including mRNA molecules;

providing a second nucleic acid sample including mRNA molecules;

contacting each of said first nucleic acid sample and said second nucleic acid sample with a first oligodeoxynucleotide primer that hybridizes to a first site in mRNAs in said first and second nucleic acid samples;

reverse transcribing said mRNAs to which said first primer hybridizes to produce a first population of DNA strands that are complementary to said mRNAs in said first nucleic acid sample to which said first primer hybridizes, and a second population of DNA strands that are complementary to said mRNAs in said second nucleic acid sample to which said first primer hybridizes;

contacting said first and second populations of DNA strands with a second oligodeoxynucleotide primer that hybridizes to a second site in said first and second populations of DNA strands, which second site includes NNNRNNATGN (SEQ ID NO;

27), said contacting being performed under conditions in which said second primer hybridizes with at least some of the DNA strands in said first and second populations;

extending said second primer to produce a third population of DNA strands that are complementary to the DNA strands in said first population to which said second primer hybridizes, and a fourth population of DNA strands that are complementary to the DNA strands in said second population to which said second primer hybridizes;

amplifying portions of the DNA strands in said first and third populations of DNA strands with said first and second primers to produce a first population of amplification products;

amplifying portions of the DNA strands in said second and fourth populations of DNA strands with said first and second primers to produce a second population of amplification products;

comparing the presence or level of individual amplification products in said first and second populations of amplification products.

2. The method of claim 1 wherein said first nucleic acid sample comprises mRNAs expressed in a first cell and said second nucleic acid sample consists of mRNAs expressed in a second cell.

3. The method of claim 1 wherein said first nucleic acid sample comprises mRNAs expressed in a cell at a first developmental stage and said second nucleic acid sample comprises mRNAs expressed in said cell at a second developmental stage.

4. The method of claim 1 wherein said first nucleic acid sample comprises mRNAs expressed in a tumorigenic cell and said second nucleic acid sample comprises mRNAs expressed in a normal cell.

5. The method of claim 1 wherein said first primer has an arbitrary nucleotide sequence and is at least 6 nucleotides in length.

6. The method of claim 5 wherein said first primer is about 9-10 nucleotides in length.

7. The method of claim 5 wherein said first primer has a GC content within the range of about 50-70%.

8. The method of claim 1 wherein the nucleotide sequence of said first or said second primer includes a sequence that is recognized by a restriction endonuclease.

9. The method of claim 1 wherein the nucleotide sequence of said first primer includes a sequence substantially complementary to a consensus sequence found in a gene family.

10. The method of claim 1 wherein the nucleotide sequence of said first primer includes a sequence substantially complementary to a sequence found in an mRNA of known sequence.

11. The method of claim 1 wherein at least one of said first and second primers comprises a plurality of oligodeoxynucleotides.

12. The method of claim 1 wherein individual oligodeoxynucleotide molecules in said plurality of oligodeoxynucleotides have the same nucleotide sequence.

13. The method of claim 1 wherein individual oligodeoxynucleotide molecules in said plurality of oligodeoxynucleotides have different nucleotide sequences.

14. The method of claim 1 wherein the amplifying steps each comprise performing a polymerase chain reaction in which the concentration of dNTPs is at or below approximately 20 μ

M.

15. The method of claim 1 wherein the amplifying steps each comprise performing a polymerase chain reaction in which the concentration of dNTPs is approximately 2 μ

M.

16. The method of claim 1 wherein said first and second sites are separated from one another so that at least some amplification products in said first and second populations of amplification products have a size in the range of approximately 100-500 basepairs.

17. The method of claim 1 wherein the step of comparing comprises resolving each of said first and second populations of amplification products by gel electrophoresis and comparing the presence or level of bands of particular sizes.

18. The method of claim 1 further comprising a step of cloning individual amplification products from said first or second populations of amplification products.

19. The method of claim 1 further comprising a step of detecting a difference in the presence or level of an individual amplification product in said first population of amplification products as compared with said second population of amplification products.

20. The method of claim 19 further comprising a step of isolating an individual amplification product whose presence or level differs in said first and second populations of amplification products.

21. The method of claim 20 further comprising a step of cloning said isolated amplification product into a vector.

22. The method of claim 20 or 21 further comprising a step of screening a nucleic acid library with said isolated amplification product.

23. The method of claim 20 or 21 further comprising a step of determining the nucleotide sequence of at least a portion of said isolated amplification product.