cDNA library preparation
First Claim
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1. A method for generating a library from RNA comprising the steps of:
- (a) fragmenting said RNA to produce fragmented RNAs;
(b) hybridizing a plurality of primers to said fragmented RNAs to form hybridized primers;
(c) elongating said hybridized primers with reverse transcriptase to form a plurality of single stranded cDNAs from said RNA, wherein said single stranded cDNAs comprises said plurality of primers at a 5′
end;
(d) ligating a first adaptor to said 5′
end of said cDNA, wherein said adaptor comprises an overhanging 5′
end region which is complementary to a 5′
end of said single stranded cDNA and ligating a second adaptor comprising an overhanging 3′
end region that is complementary to a 3′
end of said cDNA to form a single stranded cDNA comprising a first adaptor at a 5′
end and a second adaptor at a 3′
end (e) purifying said single stranded cDNAs to generate said cDNA library.
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Abstract
New biochemical protocols for high throughput processing of mRNA samples into cDNA libraries with adaptor sequences compatible with automated sequencing systems are provided. The provided methods produces cDNA libraries which do not have 3′ bias associated with current cDNA library production methods. New methods for the production of DNA libraries from DNA are also provided.
66 Citations
56 Claims
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1. A method for generating a library from RNA comprising the steps of:
-
(a) fragmenting said RNA to produce fragmented RNAs;
(b) hybridizing a plurality of primers to said fragmented RNAs to form hybridized primers;
(c) elongating said hybridized primers with reverse transcriptase to form a plurality of single stranded cDNAs from said RNA, wherein said single stranded cDNAs comprises said plurality of primers at a 5′
end;
(d) ligating a first adaptor to said 5′
end of said cDNA, wherein said adaptor comprises an overhanging 5′
end region which is complementary to a 5′
end of said single stranded cDNA and ligating a second adaptor comprising an overhanging 3′
end region that is complementary to a 3′
end of said cDNA to form a single stranded cDNA comprising a first adaptor at a 5′
end and a second adaptor at a 3′
end(e) purifying said single stranded cDNAs to generate said cDNA library. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 41)
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30. A method for generating a library from RNA comprising the steps of:
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(a) fragmenting said RNA to produce fragmented RNAs;
(b) hybridizing a plurality of primers to said fragmented RNAs to form hybridized primers wherein said primers comprise a 5′
region with an adaptor sequence and a 3′
region for hybridizing to said fragmented RNA;
(c) elongating said hybridized primers with reverse transcriptase to form a plurality of single stranded cDNAs from said RNA, wherein said single stranded cDNAs comprises said plurality of primers at a 5′
end;
(d) ligating an adaptor comprising an overhanging 3′
end region that is complementary to a 3′
end of said cDNA to form a single stranded cDNA comprising an adaptor at a 3′
end(e) purifying said single stranded cDNAs to generate said cDNA library. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
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Current Assignee454 Life Sciences Corporation (Roche Holding AG)
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Original Assignee454 Life Sciences Corporation (Roche Holding AG)
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InventorsWilloughby, David, Hutchison, Stephen, Simons, Jan
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Application NumberUS11/523,120Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12N 15/1096 cDNA Synthesis; Subtracted ...
