Direct sequencing of enzymatically amplified human genomic DNA.

Abstract

The polymerase chain reaction is a recently described technique that uses flanking oligonucleotide primers and repeated cycles of enzymatic primer extension to amplify a short segment of DNA by >100,000-fold. By use of sequencing primers located internal to the amplification primers, direct genomic sequence was obtained from enzymatically amplified DNA by using the dideoxynucleotide chaintermination method. The method is relatively simple and offers significant advantages in identifying mutations in genes for which the normal sequence is known. Heterozygous and homozygous mutations in the human A-and y-globin loci were unambiguously identified in 3 days with <1 pg of genomic DNA.The identification of mutations and polymorphisms in human genes by DNA sequencing contributes substantially to our understanding of the molecular nature of disease and has a variety of practical applications in diagnosis. Sequence information is usually obtained by cloning the genes from individuals expected to harbor a mutation and then comparing the sequence of the cloned DNA to that of the normal allele. The cloning process is laborious, however, requiring several weeks of library construction and screening to obtain DNA clones. Moreover, if the individual is not homozygous, several clones may have to be studied to be certain that both alleles are detected.Several methods of screening for mutations have recently been developed that do not require cloning. Carefully controlled hybridization with mutation-specific oligonucleotide probes (1), for example, can detect the presence of single base changes in short segments ofDNA if the exact nature of the mutation is known in advance. The location of mutations in a few genes has also been successfully identified by ribonuclease cleavage of mismatches between a labeled normal antisense RNA and genomic DNA (2) or by cleavage of RNARNA duplexes of a labeled antisense RNA and cellular RNA from an affected tissue (3, 4). Mutations in genomic DNA have also been detected by denaturing gradient gel electrophoresis (5). These approaches, however, do not provide the actual sequence at the mutation site, and some mismatches are not detected. Methods have been reported for direct chemical sequencing of mammalian genomic DNA (6), but these have not yet been widely adopted due to the degree of technical difficulty. Direct dideoxynucleotide sequencing (7) of eukaryotic genomic DNA has previously only been achieved in yeast (8).The development of the polymerase chain reaction (PCR; ref. 9) to selectively amplify a short segment of DNA by >100,000-fold has reduced the problem of sequence complexity and signal strength as impediments to direct analysis of single-copy DNA sequences. The PCR has been used to enrich for segments of the human f3-globin (9-11), HLADQa (10, 11), and c-ras (12) genes and has recently been used to acquire direct sequence from human mitochondrial DNA (12). The misincorporation rate of PCR DNA synthesis is low [1 in 600 base pairs (bp) or less], but in a cloning study only 1-2...

Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in ten subjects determined by direct sequencing of amplified transcripts.

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Phenotypes and phosphatidylinositol glycan-class A gene abnormalities during cell differentiation and maturation from precursor cells to mature granulocytes in patients with paroxysmal nocturnal hemoglobinuria

“…The frequencies of mutant PIG-A gene clones detected only once were 102 (14.6%) of 700 clones from granulocytes and 80 (12.9%) of 621 clones from CD34 ϩ cells. Also, we sequenced PCR-amplified DNA 39,40 from CD34 ϩ CD59 Ϫ cells and CD59 Ϫ and CD59 ϩ/Ϫ granulocytes from case 1, CD34 ϩ CD59 Ϫ cells and CD59 Ϫ granulocytes from case 2, and CD34 ϩ CD59 Ϫ cells and CD59 Ϫ granulocytes from case 3. CD59 ϩ granulocytes from the PNH patients and a healthy volunteer were used as controls.…”

Demonstration of human Borna disease virus RNA in human peripheral blood mononuclear cells

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