Complementary DNAs
a technology of complementary dnas and dnas, applied in the field of complementary dnas, can solve the problems of requiring years of effort, affecting the purification effect of desired proteins, and even a single human gene characterization process was a painstaking process, so as to facilitate the purification of desired proteins
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example 1
Ligation of the Nucleoside Diphosphate pCp to the 3′ End of Messenger RNA
[0077] 1 μg of RNA was incubated in a final reaction medium of 10 μl in the presence of 5 U of T4 phage RNA ligase in the buffer provided by the manufacturer (Gibco-BRL), 40 U of the RNase inhibitor RNASIN (Promega) and, 2 μl of 32 pCp (Amersham #PB 10208).
[0078] The incubation was performed at 37° C. for 2 hours or overnight at 7-8° C.
[0079] Following modification or elimination of the 2′,3′-cis diol at the 3′ ribose, the 2′,3′-cis diol present at the 5′ end of the mRNA may be oxidized using reagents such as NaBH4, NaBH3CN, or sodium periodate, thereby converting the 2′,3′-cis diol to a dialdehyde. Example 2 describes the oxidation of the 2′,3′-cis diol at the 5′ end of the mRNA with sodium periodate.
example 2
Oxidation of 2′,3′-cis Diol at the 5′ End of the mRNA
[0080] 0.1 OD unit of either a capped oligoribonucleotide of 47 nucleotides (including the cap) or an uncapped oligoribonucleotide of 46 nucleotides were treated as follows. The oligoribonucleotides were produced by in vitro transcription using the transcription kit AMPLISCRIBE T7 (Epicentre Technologies). As indicated below, the DNA template for the RNA transcript contained a single cytosine. To synthesize the uncapped RNA, all four NTPs were included in the in vitro transcription reaction. To obtain the capped RNA, GTP was replaced by an analogue of the cap, m7G(5′)ppp(5′)G. This compound, recognized by polymerase, was incorporated into the 5′ end of the nascent transcript during the step of initiation of transcription but was not capable of incorporation during the extension step. Consequently, the resulting RNA contained a cap at its 5′ end. The sequences of the oligoribonucleotides produced by the in vitro transcription reac...
example 3
Coupling of the Dialdehyde with Biotin
[0083] The oxidation product obtained in Example 2 was dissolved in 50 μl of sodium acetate at a pH of between 5 and 5.2 and 50 μl of freshly prepared 0.02 M solution of biotin hydrazide in a methoxyethanol / water mixture (1:1) of formula:
[0084] In the compound used in these experiments, n=5. However, it will be appreciated that other commercially available hydrazides may also be used, such as molecules of the formula above in which n varies from 0 to 5.
[0085] The mixture was then incubated for 2 hours at 37° C. Following the incubation, the mixture was precipitated with ethanol and dialyzed against distilled water.
[0086] Example 4 demonstrates the specificity of the biotinylation reaction.
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