Method for synthesis of the second strand of cDNA
Synthesis of the second strand of cDNA using pol ∝-primase is provided. Pol ∝-primase is used to initiate primers on a cDNA strand de novo, followed by extension of the primer with pol ∝-primase, a second DNA polymerase or a combination thereof. In a preferred embodiment, pol ∝-primase extends the primer, followed by addition of a second DNA polymerase, preferably Klenow fragment.
1. A method for preparing double stranded cDNA comprising the steps of:
a) synthesizing a first cDNA strand using an RNA strand as a template to obtain a cDNA:RNA hybrid;
b) separating and purifying the first cDNA strand from the RNA strand;
c) contacting the first cDNA strand with pol α-primase, wherein the pol α-primase is at a concentration effective for sequence-independent initiation, and wherein the pol α-primase synthesizes an RNA primer using the first cDNA strand as a template; and
d) extending the RNA primer from about 30 to about 70 nucleotides with the pol α-primase, followed by addition of a second DNA polymerase to generate a full length double stranded cDNA, using the first cDNA strand as a template.
2. The method of claim 1 , wherein the second DNA polymerase is Klenow fragment.
3. A process for preparing a double stranded cDNA comprising the steps of:
a) separating an RNA strand from a DNA strand of a double stranded cDNA:RNA hybrid to form a single stranded cDNA;
b) contacting the separated cDNA strand with a pol α-primase, wherein the pol α-primase primase is at a concentration effective for sequence-independent initiation, and wherein the pol α-primase synthesizes an RNA primer using the separated cDNA strand as a template; and
c) extending the RNA primer from about 30 to about 70 nucleotides with the pol α-primase, followed by addition of a second DNA polymerase to generate a full length double stranded cDNA, using the cDNA as a template.
4. The method of claim 3 , wherein the second DNA polymerase is Klenow fragment.
5. A process for preparing a double stranded cDNA comprising synthesizing a primer with pol α-primase on a purified cDNA strand, wherein the pol α-primase is present in a concentration effective for sequence-independent initiation, followed by primer extension from about 30 to about 70 nucleotides with the pol α-primase, followed by further extension with a second DNA polymerase to obtain a full length double stranded cDNA.
6. The method of claim 5 , wherein the second DNA polymerase is Klenow fragment.
7. The method of claim 5 , wherein the double stranded cDNA is about 1000 nucleotides.
8. The method of claim 1 , wherein the double stranded cDNA is about 1000 nucleotides.
9. The method of claim 3 , wherein the double stranded cDNA is about 1000 nucleotides.
10. The method of claim 3 , wherein the first cDNA strand is separated from the RNA strand by enzymatically degrading the RNA strand.
11. The method of claim 10 , wherein the first cDNA strand is purified.
12. A process for preparing a full length double stranded cDNA comprising the steps of:
a) enzymatically transcribing a first DNA strand from an RNA strand to form a DNA:RNA duplex;
b) isolating the DNA strand from the RNA strand; and
c) synthesizing a second DNA strand with pol α-primase in combination with another polymerase using the first DNA strand as a template to form full length double stranded cDNA of about 1000 nucleotides,
wherein the second DNA strand is synthesized by synthesizing a primer with pol α-primase, followed by primer extension from about 30 to about 70 nucleotides with the pol α-primase, followed by further extension with a second DNA polymerase,
wherein the pol α-primase is present in a concentration effective for sequence-independent initiation.