US 5,665,547 AGranted

Methods of comparing levels or amounts of mRNAs

AssigneeDana-Farber Cancer Institute, Inc.

InventorsArthur B. Pardee, Peng Liang

Life of this patent

Estimating the term…

Loading patent…

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.

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.

6 more elements

Facts

Application

08/430,536Filed 25 Apr 1995 · continuation

Priority

11 Mar 1992

Granted

9 Sep 1997

Term ends

Not estimatedThe source records don’t support an estimate.

Classification8 codes

C12N 15/1096
Processes for the isolation, preparation or purification of DNA or RNA: cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
C12N 15/101
By means of a solid support carrier, e.g. particles, polymers: by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
C12Q 1/6809
Involving nucleic acids: methods for determination or identification of nucleic acids involving differential detection
C12Q 1/686
Nucleic acid amplification reactions: polymerase chain reaction [PCR]

Family

Serial850,343Parent of this continuation-in-part · filed 11 Mar 1992

Serial33,084Parent of this continuation · filed 11 Mar 1993

Serial351,748Parent of this continuation · filed 8 Dec 1994

US 5,262,311 AParent of this continuation-in-part

US 5,665,547 AThis patent

From the US filing history. Foreign family members aren’t listed.

Abstract

Claims24