TY - JOUR
T1 - An Improved Method to Obtain a Large Number of Mutants in a Defined Region of DNA
AU - Pine, Richard
AU - Huang, P. C.
N1 - Funding Information:
Work was supported in part by the National Institutes of Health, Grant R01 GM 32606. R.P. is an NIA postdoctoral trainee.
PY - 1987/1
Y1 - 1987/1
N2 - This chapter discusses the improved method to obtain a large number of mutants in a defined region of deoxyribo nucleic acid (DNA). Conditions for mutagenesis of nucleic acids with sodium bisulfite have been developed, based on the well-characterized reactions of this reagent with the bases of nucleosides, nucleotides, and nucleic acids. The reaction with uracil and thymine is fully reversible, while the adduct of cytosine can undergo hydrolytic deamination to produce 5,6-dihydrouracil-6-sulfonate. An approach in which large numbers of altered molecules are produced with random mutations in single or multiple occurrences is required for certain targets. Such a method can be designed by adaptation of low frequency mutagenesis with sodium bisulfite in combination with appropriate cloning and screening. In addition to the criteria, on which other uses of random mutagenesis are based, these targets should be sequences with many putatively important sites. Mutants containing varied but specific base alterations throughout a gene would provide a sufficient choice of replacements to be compared and studied.
AB - This chapter discusses the improved method to obtain a large number of mutants in a defined region of deoxyribo nucleic acid (DNA). Conditions for mutagenesis of nucleic acids with sodium bisulfite have been developed, based on the well-characterized reactions of this reagent with the bases of nucleosides, nucleotides, and nucleic acids. The reaction with uracil and thymine is fully reversible, while the adduct of cytosine can undergo hydrolytic deamination to produce 5,6-dihydrouracil-6-sulfonate. An approach in which large numbers of altered molecules are produced with random mutations in single or multiple occurrences is required for certain targets. Such a method can be designed by adaptation of low frequency mutagenesis with sodium bisulfite in combination with appropriate cloning and screening. In addition to the criteria, on which other uses of random mutagenesis are based, these targets should be sequences with many putatively important sites. Mutants containing varied but specific base alterations throughout a gene would provide a sufficient choice of replacements to be compared and studied.
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U2 - 10.1016/0076-6879(87)54088-5
DO - 10.1016/0076-6879(87)54088-5
M3 - Article
C2 - 3323815
AN - SCOPUS:0023478077
VL - 154
SP - 415
EP - 430
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
IS - C
ER -