Proportional amplification of mRNA from a linear template in vitro

• US 6,203,984 B1
• Filed: 07/02/1998
• Issued: 03/20/2001
• Est. Priority Date: 07/02/1998
• Status: Expired due to Fees

• Current Assignee: Pacron, Inc.
• Original Assignee: Pacron, Inc.
• Inventors: Peng, Allan, Hu, Qianjin
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/109,377
• Time in Patent Office: 992 Days
• Field of Search: 435/6, 435/69.1, 435/320.1, 435/91.1, 435/91.2
• 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 2521/501 Ligase
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2539/101 Subtraction analysis

Abstract

The invention concerns polynucleotide compositions and methods for copying and amplifying mRNA, and provides for a method of making a linear cDNA or cRNA library based on original mRNA templates. The linear polynucleotides have the advantage of being more completely representative of full length mRNA transcripts than previously possible, and the linear libraries constructed by these methods represent relative abundances of the original transcripts in the original population of mRNA more accurately than previously possible.

Claims

1. A method of forming a DNA template for producing mRNA comprising the steps of:

providing a single stranded DNA molecule comprising in 5′

to 3′

order;

(a) a promoter, (b) a restriction endonuclease cleavage site, (c) an oligonucleotide dT sequence having at least 10 dTs, and (d) a coding sequence, wherein the promoter is oriented such 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);

ligating the single-stranded DNA molecule to form a linear DNA catenate comprising at least two series of elements (a), (b), (c), and (d) or a circular DNA;

providing a second strand complementary to and hydrogen-bonded to the single stranded DNA molecule to form a double-stranded linear catenate or a circular DNA molecule; and

cleaving the double-stranded linear catenate or the circular DNA molecule with the restriction endonuclease to form the DNA template having a promoter upstream of the coding sequence wherein the promoter is oriented to transcribe downstream.

2. A method of forming a DNA template for producing antisense mRNA comprising the steps of:

providing a single stranded DNA molecule comprising in 5′

to 3′

order;

(a) a restriction endonuclease cleavage site, (b) a promoter, (c) an oligonucleotide dT sequence having at least 10 dTs, and (d) a first cDNA sequence, wherein the promoter is oriented such 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);

ligating the single-stranded DNA molecule to form a linear DNA catenate comprising at least two series of elements (a), (b), (c), and (d) or a circular DNA;

providing a second strand complementary to and hydrogen-bonded to the single stranded DNA molecule to form a double-stranded linear catenate or a circular DNA molecule; and

cleaving the double-stranded linear catenate or a circular DNA molecule with the restriction endonuclease to form the DNA template having the promoter downstream of the coding sequence wherein the promoter is oriented to transcribe upstream.

3. 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, deoxyribonucleotide triphosphates, 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.

4. The method of claim 3, further comprising,

(g) transcribing the double stranded template of step (f) with RNA polymerase and nucleotide triphosphates, to form single stranded cRNA.

5. The method of claim 3, further comprising,

(h) amplifying the double stranded template of step (f) with DNA polymerase, deoxyribonucleotide triphosphates, 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.

6. The method of claim 3, further comprising

(i) transcribing and translating in vitro the double stranded template of step (f) to make a polypeptide encoded by the cDNA.

7. The method of claim 3, wherein step (b) is performed using RNase or NaOH.

8. The method of claim 3, wherein the promoter is selected from the group consisting of a T7, T3 and SP6 promoter.

9. The method of claim 3, wherein the mRNA is present in a preparation of total RNA.

10. The method of claim 3, wherein the restriction endonuclease site comprises 8 nucleotides.

13. The method of claim 10, wherein the second oligonucleotide primer further comprises a second promoter upstream of the oligonucleotide dT sequence, wherein the promoter is oriented such that it initiates transcription downstream and further wherein the second promoter comprises a promoter sequence distinct from the first promoter sequence.

14. The method of claim 10, further comprising

(f) transcribing the double stranded cDNA of step (e) with RNA polymerase, nucleotide triphosphates, and a primer sequence comprising a promoter, to form cRNA.

11. A method of making cDNA comprising,

(a) decapping an mRNA,

(b) ligating a first oligonucleotide primer comprising a first promoter to a 5′

end of the mRNA molecule to form an RNA template, wherein the first promoter is oriented to transcribe towards the 3′

end,

(c) reverse transcribing the RNA template of step (b) with reverse transcriptase, deoxyribonucleotide triphosphates 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, deoxyribonucleotide triphosphates, and a third oligonucleotide primer comprising at least 12 nucleotides of the first primer sequence, to form double stranded cDNA.

12. A method of making cDNA comprising,

(a) decapping an mRNA,

(b) ligating a first oligonucleotide primer to a 5′

end of the mRNA molecule to form an RNA template,

(c) reverse transcribing the RNA template of step (b) with reverse transcriptase, deoxyribonucleotide triphosphates and a second oligonucleotide primer comprising an oligonucleotide dT sequence of at least 10 dTs, and a promoter upstream of the oligonucleotide dT sequence, wherein the promoter is oriented so that it initiates transcription downstream, to form first strand cDNA,

(d) removing the mRNA from the first strand cDNA,

(e) incubating the first strand cDNA, a DNA polymerase, deoxyribonucleotide triphosphates, and a third oligonucleotide primer comprising at least 12 nucleotides of the first primer sequence, to form double stranded cDNA.

15. A method of subtractive hybridization identifying mRNA transcripts that are not common to two populations of mRNA comprising,

(a) reverse transcribing a first mRNA population with reverse transcriptase, deoxyribonucleotide triphosphates, 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 (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 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, deoxyribonucleotide triphosphates, 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.

16. 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, deoxyribonucleotide triphosphates, 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 (a) to the mRNA of step (a) to form a composition comprising cDNA;

mRNA hybrids of some but not all of the cDNA and mRNA pairs that can hybridize,

(d) eliminating the cDNA;

mRNA hybrids that are formed 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, deoxyribonucleotide triphosphates, 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 that are not common to the two mRNA populations.

17. The method of claim 16, wherein step (c) comprises an incubation ranging from 2 hours to 48 hours.