Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-02
2001-03-20
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091100, C435S091200, C435S320100
Reexamination Certificate
active
06203984
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to compositions and methods for amplification of mRNA.
BACKGROUND
Standard procedures for copying mRNA and amplifying mRNA exist in the art, and are the foundation of much of the recent work and advancements in molecular biology. Copying and amplifying mRNA, and using these copies to generate a library of polynucleotides that represent the original mRNA provides researchers with templates for cloning and analysis of the original transcripts. Knowledge of the original transcripts allows analysis and manipulation of the expression products of the genes upon which the transcripts are based. Any tools that can further the usefulness and accuracy of methods of copying and amplifying mRNA are extremely useful and sought after in the fields of molecular biology and functional genomics.
SUMMARY
The invention is a single stranded DNA molecule comprising in 5′ to 3′ order: (a) a promoter, (b) a restriction endonuclease cleavage site, and (c) an oligonucleotide dT sequence having at least 10 dTs, wherein the promoter is oriented so that it initiates transcription towards the 5′ end, and further wherein between 0 and 100 nucleotides are between each of elements (a), (b), and (c). This molecule can further comprise additional nucleotides 3′ of the oligonucleotide dT sequence, and these additional nucleotides can comprise a first strand cDNA sequence. The molecule can be circular, or a linear catenate of at least two series of elements (a), (b), (c), and (d). These molecules can further comprise a second strand complementary to and hydrogen-bonded to the single stranded DNA molecule. Consistent with the composition of the invention is a method of forming a template for producing mRNA comprising cleaving the DNA molecule with a second strand complementary to it with the restriction endonuclease to form a DNA template having a promoter upstream of a coding sequence oriented to transcribe downstream.
Similarly, for the purpose of making antisense copies, the invention is also a single stranded DNA molecule comprising in 5′ to 3′ order: (a) a restriction endonuclease cleavage site, (b) a promoter, and (c) an oligonucleotide dT sequence having at least 10 dTs, wherein the promoter is oriented so that it initiates transcription towards the 3′ end, and further wherein between 0 and 100 nucleotides are between each of elements (a), (b), and (c). For making antisense polynucleotides, the permutations described above also apply.
The invention includes a method of making cDNA comprising (a) incubating mRNA with reverse transcriptase, deoxyribonucleotide triphosphates and a first oligonucleotide primer comprising in a 5′ to 3′ orientation (i) a promoter, (ii) a restriction endonuclease cleavage site, and (iii) an oligonucleotide dT sequence having at least 10 dTs, wherein the promoter is oriented so that it initiates transcription towards the 5′ end, and further wherein between 0 and 100 nucleotides are between each of elements (i), (ii), and (iii) to form first strand cDNA comprising at its 5′ end the first oligonucleotide primer, (b) removing the mRNA from the first strand cDNA, (c) phosphorylating the 5′ end of the first strand cDNA, (d) ligating the phosphorylated first strand cDNA with ligase to form a linear catenate of first strand cDNA or a circular first strand cDNA, (e) incubating the linear catenate or circular cDNA with a DNA polymerase, deoxyribonucleotidetriphosphates, and a second oligonucleotide primer which is complementary to at least 12 nucleotides of the first oligonucleotide primer to form a double stranded linear catenate or circular cDNA, (f) cleaving the double stranded linear catenate or circular cDNA with the restriction endonuclease to form a double stranded template having the promoter upstream of the cDNA, oriented to transcribe downstream. This method can further comprise step (g) transcribing the double stranded template of step (f) with RNA polymerase and nucleotidetriphosphates to form single stranded cRNA. Still further the method can comprise step (h) amplifying the double stranded template of step (f) with DNA polymerase, deoxyribonucleotidetriphosphates, a third oligonucleotide primer complementary to at least 12 nucleotides of the 3′ end of a first strand of the cDNA, and a fourth oligonucleotide primer complementary to at least 12 nucleotides of the 3′ end of the second strand. Still further the method can comprise step (i) transcribing and translating in vitro the double stranded template of step (f) to make a polypeptide encoded by the cDNA.
Another method of the invention is a method of making cDNA comprising, (a) decapping an mRNA, (b) ligating a first oligonucleotide primer comprising a promoter to a 5′ end of the mRNA molecule to form an RNA template, wherein the promoter is oriented to transcribe towards the 3′ end, (c) reverse transcribing the RNA template of step (b) with reverse transcriptase, deoxyribonucleotidetriphosphates and a second oligonucleotide primer comprising an oligonucleotide dT sequence of at least 10 dTs, and an additional nucleotide sequence 5′ of the oligonucleotide dT sequence, to form first strand cDNA, (d) removing the mRNA from the first strand cDNA, (e) incubating the first strand cDNA, a DNA polymerase, deoxyribonucleotidetriphosphates, and a third oligonucleotide primer comprising at least 12 nucleotides of the first primer sequence, to form double stranded cDNA.
Further the invention includes a method of substractive hybridization identifying mRNA transcripts that are not common to two populations of mRNA comprising, (a) reverse transcribing a first mRNA population with reverse transcriptase, deoxyribonucleotidetriphosphates, and a first nucleotide primer comprising in 5′ to 3′ order: (i) a promoter, (ii) a restriction endonuclease cleavage site, and (iii) an oligonucleotide dT sequence having at least 10 dTs, wherein the promoter is oriented so that it initiates transcription towards the 5′ end, and further wherein between 0 and 100 nucleotides are between each of elements (i), (ii), and (ii), to form first strand cDNA comprising at a 5′ end the first primer sequence, (b) removing the mRNA from the first strand cDNA, (c) hybridizing the first strand cDNA of step (b) to a second mRNA population to form a composition comprising cDNA:mRNA hybrids and nonhybridized first strand cDNA, (d) eliminating the cDNA:mRNA hybrids from the composition, (e) ligating the composition comprising nonhybridized first strand cDNA with ligase to form linear catenates or circular first strand cDNA, (f) incubating the first strand linear catenates or circular cDNA with DNA polymerase, deoxyribonucleotidetriphosphates, and a second oligonucleotide primer complementary to at least 12 nucleotides of the first primer sequence to form double stranded linear catenates or circular cDNA, (g) cleaving the double stranded linear catenates or circular cDNA with the restriction endonuclease to form cDNA having a promoter upstream of a coding sequence oriented to transcribe downstream representing mRNA molecules which are not common to the two mRNA populations.
The invention also includes a method of reducing the quantity of a select mRNA present in a population of mRNA comprising, (a) reverse transcribing a first mRNA population with reverse transcriptase, deoxyribonucleotidetriphosphates, and a first nucleotide primer comprising in 5′ to 3′ order: (i) a promoter, (ii) a restriction endonuclease cleavage site, and (iii) an oligonucleotide dT sequence having at least 10 dTs, wherein the promoter sequence is oriented so that it initiates transcription towards the 5′ end, and further wherein between 0 and 100 nucleotides are between each of elements (i), (ii), and (iii), to form first strand cDNA comprising at a 5′ end the first primer sequence, (b) removing the mRNA from the first strand cDNA, (c) hybridizing for a sufficient period of time the first strand cDNA of step (
Hu Qianjin
Peng Allan
Marschel Ardin H.
Morrison & Foerster / LLP
Pacron, Inc.
LandOfFree
Proportional amplification of mRNA from a linear template in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Proportional amplification of mRNA from a linear template in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Proportional amplification of mRNA from a linear template in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2488986