TY - JOUR
T1 - Different restriction enzyme-generated sticky DNA ends can be joined in vitro
AU - Hung, Mien Chie
AU - Wensink, Pieter C.
N1 - Funding Information:
ACKNOWLEDGEMENTS We thank Robert Schleif, Howard Baum and the two anonymous referees for very useful criticism of this manuscript. This work was supported by a research grant (GM21626) from the National Institutes of Health.
PY - 1984/2/24
Y1 - 1984/2/24
N2 - We describe a simple method for joininq the 5′-protruding, single-stranded DNA ends generated by restriction enzymes. The method allows ends with different sequences to be joined and prevents identical ends from being joined. This is accomplished by partially fillinq the single strands in a controlled reverse transcriptase reaction. Partial filling can create new single-stranded ends that can be ligated to different, partially filled ends. In almost all useful cases, partial filling simultaneously eliminates the self-complementarity of identical ends and thus prevents them from being joined by DNA ligase. Althouoh all possible combinations of partially filled ends were not tested, the tests performed indicate that the method is fairly general. We demonstrate that ends of the same length can be ligated with useful efficiency if they are: 1) one nucleotide long and complementary; 2) two nucleotides long and complementary or have a mismatch (dA:dC) at one position; or 3) three nucleotides long and, in our test, have a dT:dC mismatch at the middle position.
AB - We describe a simple method for joininq the 5′-protruding, single-stranded DNA ends generated by restriction enzymes. The method allows ends with different sequences to be joined and prevents identical ends from being joined. This is accomplished by partially fillinq the single strands in a controlled reverse transcriptase reaction. Partial filling can create new single-stranded ends that can be ligated to different, partially filled ends. In almost all useful cases, partial filling simultaneously eliminates the self-complementarity of identical ends and thus prevents them from being joined by DNA ligase. Althouoh all possible combinations of partially filled ends were not tested, the tests performed indicate that the method is fairly general. We demonstrate that ends of the same length can be ligated with useful efficiency if they are: 1) one nucleotide long and complementary; 2) two nucleotides long and complementary or have a mismatch (dA:dC) at one position; or 3) three nucleotides long and, in our test, have a dT:dC mismatch at the middle position.
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U2 - 10.1093/nar/12.4.1863
DO - 10.1093/nar/12.4.1863
M3 - Article
C2 - 6199744
AN - SCOPUS:0021769623
SN - 0305-1048
VL - 12
SP - 1863
EP - 1874
JO - Nucleic acids research
JF - Nucleic acids research
IS - 4
ER -