Showing papers by "Henry A. Erlich published in 1987"

Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme

Abstract: A process for amplifying any target nucleic acid sequence contained in a nucleic acid or mixture thereof comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers and extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The amplified sequence can be readily detected. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed.

Characterization of β-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA

Abstract: Direct sequencing of specific regions of genomic DNA1 became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA2–5. Recently, human mitochondria! DNA was amplified and directly sequenced6. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R. K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with β-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106–107 and the other is an A–C transversion at the cap site (+1) of the β-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.

Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA

Abstract: A specific segment of mitochondrial DNA from 18 people was examined by two methods of direct DNA sequencing. This segment includes a small noncoding region (V) shown before by restriction analysis to exhibit length polymorphism. All 11 of the human mtDNAs previously reported to have a deletion in this region proved to lack one of the two adjacent copies of a 9-base-pair sequence normally present in human mtDNAs. Phylogenetic analysis suggests that this deletion occurred only once during the evolution of modern types of human mtDNA and that it will be a valuable anthropological marker for peoples of East Asian origin. The one human mtDNA reported to have an addition in region V differs from the wild type by two mutations in the first copy of the 9-base-pair sequence: one transition and an addition of four cytosines, thereby producing a run of 11 cytosines. One of the direct DNA sequencing methods uses a single oligonucleotide primer to facilitate dideoxy sequencing from purified mtDNA templates. The second, more successful, method first amplifies this mtDNA segment enzymatically with two flanking primers (the "polymerase chain reaction") and then uses a third primer for DNA sequencing. This latter method, which works on the DNA extracted from small amounts of blood as well as on purified mtDNA, is shown to be a rapid means of defining sequence variants without purifying and cloning the same DNA segment from many individuals.