Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes.

Abstract

In this report, we describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., cystic fibrosis and sickle cell disease), and in others in which multiple mutations cause the disease and the sequence variation in an affected member of a given family is known (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5' end of the mutation site, and either an a-32P-labeled nucleotide corresponding to the normal coding sequence at the mutation site or an a-32P-labeled nucleotide corresponding to the mutant sequence. Single nucleotide primer extensions are then carried out and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. As predicted by the Watson-Crick base-pair rule, in the wild type only the normal base, in an affected member only the mutant base, and in carriers both the normal and the mutant base are incorporated into the primer. Thus, an essential feature of the present methodology is that the base immediately 3' to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.One goal of molecular biology is to identify the mutations that cause genetic diseases and to develop strategies and related technologies to diagnose them. Toward this end, in the past decade or so many methodological advances have been made to detect the human genetic abnormalities at the DNA level. These include indirect methods such as linkage analysis by the Southern blotting technique (1) in which the inheritance of a disorder is associated with the presence of a restriction fragment length polymorphism (RFLP)-e.g., Duchenne muscular dystrophy (2). Other indirect methods include RNase A cleavage at mismatches in probe RNA-sample DNA duplexes or denaturing gradient gel electrophoresis for mismatches in probe DNA-sample DNA duplexes-e.g., 3-thalassemia (3, 4). The direct methods include detection with the restriction enzymes or with the allele-specific oligonucleotide (ASO) probes-e.g., the sickle cell mutation (5, 6).A majority of the above approaches have now been combined with the polymerase chain reaction (PCR) for diagnosis of the sequence variations (7,8). Initially, the target DNA is amplified by PCR followed by analysis of the sequence variation by ASO hybridization (e.g., the sickle cell mutation; ref. 9), restriction enzyme analysis (e.g., the sickle cell mutation and a hemophilia B mutation;...

Parallel molecular genetic analysis

“…Allele-specific mutation/polymorphism detection methods include direct DNA sequence analysis, 98 reverse dot-blot with allele-specific oligonucleotide hybridization, 99 allele-specific PCR amplification, 100 oligonucleotide ligation amplification (OLA), 101 artificial introduction of restriction sites, 102 ligase chain reaction 103 and DNA minisequence analysis. [104][105][106] PCR-OLA has been applied to DNA diagnostics, 107 genetic mapping using biallelic markers 108 and YAC library screening. 109 A variation on PCR-OLA to increase sample throughput makes use of sequencecoded separation.…”

“…110,111 Limited primer-extension techniques, primer-guided nucleotide incorporation, single-nucleotide primer extension (SNPE) or solid-phase minisequencing were developed previously to detect point mutations by single-base extension of the primer at the site of the mutation. [104][105][106] These procedures use a primer designed to hybridize just 5' of the nucleotide to be tested. The primer is extended by a single dyelabeled dideoxynucleotide, thereby indicating the identity of the target nucleotide in the template.…”

Microarray-based method for detecting methylation changes of p16Ink4agene 5’-CpG islands in gastric carcinomas

“…Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence [3,4] . At present, several molecular biology methods are routinely used to determine the methylation status of a CpG island, such as Southern blot [5] , bisulfite genomic DNA sequencing [6] , restriction enzyme-PCR [7] , methylation-specific PCR (MSP) [8] , methylation-sensitive single nucleotide primer extension (MSSNuPE) [9] , electrochemistry [10] , etc. Among these, bisulfite nucleotide sequencing is a standard technique for detailed mapping of methylated cytosine residues within a gene promoter.…”

Accurate quantitation of allele-specific expression patterns by analysis of DNA melting

“…Based on the SNP, the relative expression levels have been assessed by several methods including RNase Protection Assay (Winter et al 1985) and Single Nucleotide Primer Extension assayed by radioactive nucleotide incorporation (SNuPE) (Kuppuswamy et al 1991), or by mass spectrophotometry (rcPCR) (Knight et al 2003). These methods are all technically challenging and, more importantly, limited in their ability to precisely quantitate variations in allelic expression.…”