The use of overlapping and tailed short primers in the chromosomal assignment of short cDNAs by the polymerase chain reaction.
For the PCR-based chromosomal assignment of very short cDNA fragments specifically designed primers are required. We tested primers with very short core sequences that are identical or complementary to known cDNA sequences, with or without tails at the 5' ends. The lower limit of the core length for PCR using human chromosome templates was 14 nucleotides (nt) when they have tails. The minimal length of the tail was 2 nt when it was attached to the 5' end of a 14-nt core. In the absence of a tail, 15 nt are needed for the core to act properly. The overall size of the short cDNA fragments that could be assigned was further reduced by using a pair of primers that overlap at the 3' ends. The limits of the free energy of overlap were about -1.9 kcal/mol at 45 degrees C, -2.9 kcal/mol at 50 degrees C and -4.5 kcal/mol at 55 degrees C. A combination of these features in a primer pair allowed cDNA fragments as short as 30 nt to be assigned.
Chae
K S
KS
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Murakawa
K
K
Okubo
K
K
Matsubara
K
K
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013816:Thermodynamics
D017931:DNA Primers; D018076:DNA, Complementary; D004247:DNA