Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis.
Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers.
Bernhard Nocht Institute for Tropical Medicine, Hamburg, F.R.G.
C G
CG
Meyer
E
E
Tannich
J
J
Harders
K
K
Henco
R D
RD
Horstmann
D000595:Amino Acid Sequence; D001483:Base Sequence; D004587:Electrophoresis, Agar Gel; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012689:Sequence Homology, Nucleic Acid
D006680:HLA Antigens; D015345:Oligonucleotide Probes