Methods of comparing levels or amounts of mRNAs

• US 5,665,547 A
• Filed: 04/25/1995
• Issued: 09/09/1997
• Est. Priority Date: 03/11/1992
• Status: Expired due to Term

• Current Assignee: Dana-Farber Cancer Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Liang, Peng, Pardee, Arthur B.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/430,536
• Time in Patent Office: 868 Days
• Field of Search: 435/91.1, 435/91.2, 435/6, 435/15, 435/810, 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 comparing amounts or levels mRNAs 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 portion of a polyadenosine (polyA) tract in mRNAs in said first and second nucleic acid samples and also to at least one non-polyA nucleotide immediately upstream of said polyA portion;

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 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 primer hybridizes to said polyA portion and to at least two nucleotides immediately upstream of said polyA portion.

5. The method of claim 1 wherein said first primer hybridizes to said polyA portion and to one nucleotide immediately upstream of said polyA portion.

6. The method of claim 1 wherein said first primer includes a polyA complementary region comprising at least 11 nucleotides and, immediately downstream from said polyA-complementary region, a non-polyA complementary region comprising at least one nucleotide.

7. The method of claim 6 wherein said polyA-complementary region comprises at least 11 contiguous thymidines.

8. The method of claim 6 wherein said non-polyA-complementary region comprises at least 2 contiguous nucleotides.

9. The method of claim 7 or 8 wherein said non-poly-A-complementary region comprises 3'"'"'-NV, wherein V is one of deoxyadenosine, deoxycytidine or deoxyguanosine.

10. The method of claim 6 wherein said first primer comprises at least 13 nucleotides.

11. The method of claim 1 wherein said second primer comprises at least 6 nucleotides.

12. The method of claim 1 wherein said second primer comprises at least 9 nucleotides.

13. The method of claim 10 wherein the nucleotide sequence of said second primer is selected at random.

14. The method of claim 1 wherein the nucleotide sequence of said first or said second primer contains a restriction endonuclease recognition site.

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

16. The method of claim 15 wherein said plurality of oligodeoxynucleotides comprises a plurality of oligodeoxynucleotide molecules having the same nucleotide sequence.

17. The method of claim 15 wherein individual oligodeoxynucleotide molecules in said plurality of oligodeoxynucleotides have different nucleotide sequences.

18. The method of claim 1 wherein said first oligodeoxynucleotide includes a sequence substantially identical to a sequence contained within an mRNA of known sequence.

19. The method of claim 1 further comprising the 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 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.

21. 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.

22. 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.

23. The method of claim 1 wherein said first cell comprises a tumorigenic cell and said second cell comprises a normal cell.

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