RESOLUTION OF A MISSENSE MUTANT IN HUMAN GENOMIC DNA BY DENATURING GRADIENT GEL-ELECTROPHORESIS AND DIRECT SEQUENCING USING INVITRO DNA AMPLIFICATION - HPRT MUNICH

  • 1 May 1988
    • journal article
    • research article
    • Vol. 42  (5) , 726-734
Abstract
The combination of denaturing gradient gel electrophoresis (DGGE) and in vitro DNA amplification has allowed us to (1) localize a DNA mutation to a given 100-bp region of the human genome and (2) rapidly sequence the DNA without cloning. DGGE showed that a mutation had occurred, but the technique revealed little about the nature or position of that mutation. The region of the genome containing the mutation was amplified by the polymerase chain-reaction technique, providing DNA of sufficient quality and quantity for direct sequencing. Amplification was performed with a 32P end-labeled primer that allowed direct Maxam-Gilbert sequencing of the amplified product without cloning. HPRTMunich was found to contain a single-base-pair substitution, a C-to-A transversion at base-pair position 397. We report the generation of a 169-bp, wild-type DNA probe that encompasses most of exon 3 of the human hypoxanthine guanine phosphoribosyltransferase (HRPT) gene and contains a low-temperature melting domain of .apprx. 100 bp. HPRTMunich, an HPRT mutant isolated from a patient with gout, has a single amino acid substitution; the corresponding DNA sequence alteration must lie within the low-temperature melting domain of exon 3. We report the separation of HPRTMunich from the wild-type sequence using DGGE. In addition to base-pair substitutions, DGGE is also sensitive to the methylation state of the molecule. The cDNA for HPRT was cloned into a vector and propagated in Escherichia coli dam+ and dam- strains; thus, methylated and unmethylated HRPT cDNA was obtained. A single dam methylation site exists within exon 3, and we have separated the methylated and unmethylated sequences by DGGE. To rapidly sequence HPRTMunich, we used the polymerase chain reaction. Two 20-bp olgionucleotide primers complementary to the intron sequence immediately 5'' and 3'' exon 3 were hybridized in solution to wild-type or HPRTMunich genomic DNA, and the primed template was extended with T4 DNA polymerase. The process was repeated 30 times, yielding .apprx. 150 ng of amplified product starting from 5 .mu.g of genomic DNA. A combination of DGGE and polymerase chain reaction should permit rapid identification and sequencing of base-pair-substitution mutants in human genomic DNA. Both techniques are expected to play a significant role in the analysis of base-pair mutations and in the latters'' relationship to human disease.