Methods for constructing DNA library and support carrying DNA library immobilized thereon
AssigneesNihon Parkerizing Hiroshima Co., Ltd., Toyo Kohan Co., Ltd.
InventorsKojiro Takahashi, Osamu Takai, Michifumi Tanga
Life of this patent
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Abstract
Method and apparatus for constructing a cDNA library by hybridizing mRNA with oligo (dT) on a support and treating with a reverse transcriptase to immobilized complementary DNA, or for constructing a gDNA library by ligating a double-stranded chromosomal DNA library with an oligonucleotide on a support having a restriction enzyme site and then immobilizing the gDNA library.
Claims4
A method for immobilizing a cDNA library on an original support and at least one replica support comprising:
- a. immobilizing an amino group on the surface of the original support
- b. contacting the original support with oligo (dT)n to immobilize the oligo (dT)n onto the original support, wherein n is from 15 to 30;
- c. treating the original support with a reaction solution comprising total RNA, reverse transcriptase, and nucleotides to construct cDNA from mRNA;
- d. maintaining the original support in contact with the reaction solution at 20° C. to hybridize oligo (dT)n on the original support and mRNA in the total RNA solution;
- e. treating the original support with reverse transcriptase solution and maintaining the original support in contact with reverse transcriptase to produce a cDNA library immobilized on the original support;
- f. cleaning the original support and heating the original support to dehybridize mRNA from the original support;
- and
- g. repeating steps b–f using a replica support in place of the original support in order to produce a replica cDNA library immobilized on the support.
A method for immobilizing a gDNA library on an original support and at least one replica support comprising:
- h. immobilizing an amino group on the surface of the original
- i. contacting the original support with a sense portion of oligo nucleotide having the base sequence of a target restrictive enzyme;
- j. treating the original support with restrictive enzyme to produce a support having a complete restrictive enzyme cut portion
- k. treating the original support with a reaction solution comprising purified gDNA library solution treated with restrictive enzyme, DNA ligase solution, DNA polymerase solution, and nucleotides;
- l. maintaining the original support in contact with the reaction solution at 20° C. to produce the gDNA library immobilized by DNA ligase on the support;
- m. cleaning the original support and heating the original support to dehybridize mRNA from the original support;
- n. heating the original support to dehybridize an anti-sense portion from a double stranded sense portion and eluting the anti-sense portion of the gDNA library from the support;
- and
- o. repeating steps b–f using a replica support in place of the original support in order to produce a replica gDNA library immobilized on the support.
Dependencies are inferred from the claim text.