Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences.
Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives--betaine, dimethyl sulfoxide, and 7-deaza-dGTP--was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.
Musso
Marco
M
Istituto Giannina Gaslini, Lab Genetica Molecolare, L. go G. Gaslini 5, 16148 Genova, Italy.
Bocciardi
Renata
R
Parodi
Sara
S
Ravazzolo
Roberto
R
Ceccherini
Isabella
I
D001483:Base Sequence; D001622:Betaine / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000737:chemistry*; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D020862:GC Rich Sequence; D005784:Gene Amplification; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D011401:Promoter Regions, Genetic; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics
D003848:Deoxyguanine Nucleotides; D018398:Homeodomain Proteins; C112149:LIM homeobox transcription factor 1 beta; D060850:LIM-Homeodomain Proteins; C109220:NBPhox protein; D014157:Transcription Factors; C065361:2'-deoxy-7-deazaguanosine triphosphate; D001622:Betaine; D004247:DNA; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D004121:Dimethyl Sulfoxide