The tetramethylammonium chloride method for screening of cDNA libraries using highly degenerate oligonucleotides obtained by backtranslation of amino-acid sequences.
We describe a method for screening of cDNA libraries with highly degenerate oligonucleotides using tetramethylammonium chloride (TMAC). This method is a convenient alternative to using probes generated by the polymerase chain reaction (PCR), especially when these cannot easily be made. Nylon filters were prehybridized in buffered sodium chloride and hybridized with labelled oligonucleotide in buffer containing 3 M TMAC. In TMAC the melting temperature of the oligonucleotide is independent of the G + C content, thus only depending on the length. This was confirmed by the cloning of 13 specific cDNAs with G + C contents between 27% and 61% using 15- to 20-mer oligonucleotides with a degeneracy up to 512. The method was further improved for highly degenerate oligonucleotides by testing hybridization of four 18-mer oligonucleotides, each containing one deoxyinosine (I) instead of A, G, C or T, respectively. Oligonucleotides containing I pairing with A, G or T may have slightly lower melting temperatures than those pairing with C. At practical circumstances all oligonucleotides hybridize about equally well at hybridization temperatures 10 degrees C below the irreversible melting temperature. This was further confirmed by the cloning of four cDNAs with oligonucleotides containing deoxyinosines at 3 or 4 ambiguous positions.
Honoré
B
B
Institute of Medical Biochemistry, Aarhus University, Denmark.
Madsen
P
P
Leffers
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry*; D015723:Gene Library; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D000644:Quaternary Ammonium Compounds
D018076:DNA, Complementary; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D000644:Quaternary Ammonium Compounds; C027917:tetramethylammonium