Detection Of Single Base Changes Research Articles

TILLING in Grass Species

The reverse genetics detection of single base changes in genes of interest, whether induced or endogenous, is an extremely powerful tool for functional genomics. Two major thrusts of research in the grasses are characterizing the function of all the genes in various genomes and then comparing those

A rapid one-tube PCR method for simultaneously differentiating homozygotes and heterozygotes of the Sp1 binding site polymorphism in collagen type Iα1

Rapid detection of single-base changes is fundamental to molecular medicine. PCR amplification of specific alleles (PASA) has previously been used as a rapid method of genotyping single-nucleotide changes, but one reaction is required for each allele. This paper describes a Bidirectional PASA (Bi-PASA) method, which was developed to distinguish between homozygotes and heterozygotes in one PCR reaction. The method is tested using the Sp1 polymorphism in Collagen type Ialpha1. The results demonstrate that Bi-PASA is a simple and rapid method for detecting the zygosity of the polymorphism in a single PCR reaction.

Solution-Based Scanning for Single-Base Alterations Using a Double-Stranded DNA Binding Dye and Fluorescence-Melting Profiles

DNA molecules differing by as little as a single-base substitution have traditionally been distinguished by gel electrophoresis-based methodologies that exploit differences in the sequence-specific properties of double-stranded DNA (dsDNA) such as melting temperature and secondary conformational configuration. By comparison, solution-based fluorescence methods using sequence-specific probes are limited to detecting mutations restricted to very short segments of DNA ( approximately 20 bp). We describe a solution-based fluorescence method that discriminates between wild-type and mutant sequences using a dsDNA binding dye, and interrogates a region of >200 nucleotides. This method is based on melting theory and entails fluorescence monitoring of the melting temperatures of GC-clamped amplicons subjected to gradual and progressive thermal denaturation in the presence of a constant concentration of urea. Heterozygous samples are easily identified by the lower melting temperatures of the less thermodynamically stable heteroduplex mismatches from the wild-type:mutant DNA hybrids as compared to the more stable wild-type Watson-Crick duplexes. All of the four possible sets of mismatches (A.G/T.C, T.G/A.C, G.G/C.C, and T.T/A.A) represented in 17 heterozygous mutations distributed throughout the length of 20 different amplicons (104 to 212 bp), were distinguished from the wild-type by their altered melting profiles. This methodology is advantageous in that it obviates gel electrophoresis or labeled oligonucleotide probes. Significantly, it expands the region of interrogation for detection of single-base changes using fluorescence-based methods in solution, and is amenable for automation and adaptation to high-throughput systems.

Use of DNA toolbox for the characterization of mutation scanning methods. II: evaluation of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) is one of the most commonly used methods for searching for unknown base changes (mutations). In order to characterize systematically the effects of important physical parameters on the sensitivity and specificity of SSCP, we used the DNA toolbox constructed as described in the companion paper [2]. Using this set of DNA molecules as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base, mutated to all other bases, were generated. The behavior of these constructs in manual and automated SSCP was analyzed as a function of the size, overall base content of the fragment, nature and location of the base change, and the temperature and pH of electrophoresis. Our results demonstrate that all of these variables interact to determine the rate of detection of single-base changes, with the GC content being the predominant determinant of detection sensitivity.

Overlapping PCR for bidirectional PCR amplification of specific alleles: a rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes.

Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR Amplification of Specific Alleles) is a rapid method of genotyping single-base changes, but one reaction is required for each allele. Bidirectional PASA (Bi-PASA) was developed to distinguish between homozygotes and heterozygotes in one PCR reaction by utilizing novel primer design with appropriate cycling conditions. In Bi-PASA, one of the alleles is amplified by a PASA reaction in one direction while the second allele is amplified by a PASA reaction in the opposite direction. Two outer (P and Q) and two inner allele-specific (A and B) primers are required. In heterozygotes, three segments are amplified: a segment of size AQ resulting from one allele, another segment of size PB resulting from the second allele, and a combined segment of size PQ. In homozygotes, segment PQ and either segments AQ or PB amplify. The two inner primers (A and B) contain a relatively short complementary region and a 10-nucleotide G + C-rich 5' tail. The inner primers "switch" from low-efficiency to high-efficiency amplification when genomic DNA is replaced by previously amplified template DNA. In addition, the 5' tails prevent "megapriming". The parameters for optimizing Bi-PASA were investigated in detail for common mutations in the human factor V and catechol-O-methyltransferase genes. Guidelines for optimization of Bi-PASA also were developed and tested in a prospective study. Three additional Bi-PASA assays were optimized rapidly by utilizing these guidelines. In conclusion, Bi-PASA is a simple and rapid method for detecting the zygosity of known mutations in a single PCR reaction.

Use of psoralen-coupled nucleotide primers for screening of COL4A5 mutations in Alport syndrome

Alport syndrome [1] is a genetic renal disease characterized by hematuria and varying degrees of proteinuria, sensorineural hearing loss, and less frequently ocular lesions (lenticonus, macular lesions). Diverse mutations of the COL4A5 gene encoding the alpha 5 chain of basement membrane type IV collagen have been characterized in patients with X-linked Alport syndrome. Almost every affected family harbors a unique sequence change. Therefore, the mutation search in patients with Alport syndrome depends to a great extent on sensitivity and speed of the applied methods. In addition, future clinical diagnostic applications introduce economical aspects as to which method is chosen for mutation searches. Screening for mutations in Alport syndrome usually involves PCR amplification of single exons and subsequent analysis by single strand conformation polymorphism electrophoresis (SSCP) or, with even higher reported sensitivity, by denaturing gradient gel electrophoresis (DGGE) [2—4]. These techniques are based upon the detection of altered gel mobility of mutated DNA as compared to wild-type DNA. DGGE has been reported to be suitable for DNA fragments of 100 to 400 bp in length. One drawback is that mutations only in the domain with the lowest melting point are usually detectable. To circumvent this problem, long stretches (40 bp) of GC are added to one of the primers and then integrated into the PCR product to be analyzed. The so-called GC-clamps have very high melting temperatures and confine the adjacent DNA sequence to a single, flat melting domain that is amenable for DGGE detection of single base changes. GC-clamps have been reported to greatly increase the range of detectable mutations [2]. Besides the expense of synthesizing GC-primers, technical problems may occur with optimization of PCR conditions and yield because of the asymmetry of primer sets. Recently, psoralen phosphoramidites have become commercially available for use with oligonucleotide synthesizers. DNA amplified with a primer set containing one psoralen coupled primer can be covalently crossliriked by exposure to UV light. There is some evidence in the literature that psoralen photo crosslinked DNA behaves like GC-clamped DNA [5]. In this paper, we evaluated psoralen photo crosslinking as a less costly alternative to GC-clamping when applied to mutation screening

Comparison of DGGE and CDGE in detection of single base changes in the hamster hprt and human N- ras genes

Denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) are methods based on sequence-determined melting characteristics of DNA and thus detect different types of single base changes in the amplified fragments. We have studied detection of 19 mutations in the human N- ras oncogene and 10 mutations in exon 3 of the Chinese hamster hypoxanthine-guanine phosphoribosyltransferase ( hprt) gene using GC-clamped DGGE and CDGE. After allowing formation of heteroduplexes with the corresponding wild type sequence, all the mutations separated from the wild type in at least one concentration of the denaturants used in CDGE but two of the mutations in hprt exon 3 did not show separation in any of the DGGE runs. Melting behavior of the mutant fragments was dependent, as expected, on both the type and the location of a mutation. We describe conditions allowing separation of the mutations in the fewest possible DGGE and CDGE runs.

Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay

A solid phase DNA sequencing method for non-radioactive detection of single base changes without the need for electrophoresis is presented. The concept relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (P. Nyrén, 1987, Anal. Biochem. 167, 235-238). Immobilized DNA fragments amplified with the polymerase chain reaction are used as template. A detection primer is annealed in front of the mutation and four aliquots of this mixture are incubated with DNA polymerase and one of the four different dideoxynucleotides. The presence or absence of an incorporated dideoxynucleotide is thereafter monitored by the release of inorganic pyrophosphate during the following primer extension step. We show that the concept can be used for sequencing of single bases as well as step-wise analysis of several subsequent bases.

Separation and analysis of DNA by electrophoresis.

Pulsed-field gel electrophoresis, the polymerase chain reaction and capillary electrophoresis represent major technical advances in DNA electrophoresis in the last few years. This has lead to a number of new applications, including the cloning of large DNA fragments, multiplex analysis of DNA samples, and the detection of single base changes in DNA. New fluorescent and chemiluminescent techniques promise to simplify detection methods.

Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp

We present a simple, efficient extension of denaturing gradient gel electrophoresis that allows the detection of nearly any sequence change in a defined fragment of DNA. The fragment can be obtained either by means of the polymerase chain reaction or by restriction digestion of genomic DNA. With restriction fragments of genomic DNA, sequence information is not required, and covalent modifications in genomic DNA that are lost in a PCR, such as methylation, are detectable. We describe how a GC clamp (an arbitrary, G+C-rich sequence of 30 to 60 bp) can be attached to a selected restriction fragment present in a digest of genomic DNA. The GC clamp alters the melting properties of the fragment; this change greatly increases the fraction of possible mutations that is detectable. In a 272-bp HaeIII fragment from the human β-globin gene, we were able to detect 13 of 13 mutations tested in human genomic DNA. Four additional mutations in cloned plasmids were analyzed. The data agree with a simple theoretical model for DGGE, which predicts how two fragments, differing at a single (specified) base pair, are resolved in a gradient gel as a function of running time for the gel. The calculation assists in the design of probes and gel conditions that aid in the detection of sequence changes.

Direct sequencing of the gene for Maryland/German familial amyloidotic polyneuropathy type II and genotyping by allele-specific enzymatic amplification

Direct genomic DNA sequencing has been used to characterize the mutation associated with familial amyloidotic polyneuropathy in the Maryland/German kindred. A mutation of thymine to adenine in the prealbumin (transthyretin) gene at the position corresponding to the second base of codon 58 in the prealbumin mRNA gives a histidine for leucine substitution in the plasma protein. Since the mutation does not result in a change in the restriction pattern of the prealbumin gene, a new method for the direct detection of single base changes in genomic DNA was developed using the polymerase chain reaction and an allelespecific oligonucleotide primer.

Detection of single base changes in nucleic acids.

Detection of single base changes in nucleic acids.

Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes.

Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects approximately 50% of all possible single-base changes in DNA fragments ranging from 50 to approximately 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human beta-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from less than 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.

Molecular Biology and Human Disease

This volume is a welcome attempt to inform the newcomer (student or professional) to the field, who is not ready for the jargon and assumptions of the current literature. It admirably achieves its goal and can be profitably read. This does not mean that it is perfect or that each chapter fulfills its task at the same level. Outstanding are the chapters written by one of the editors, Alexander MacLeod. However, his comment that the oligonucleotide procedures for the detection of single-base changes in DNA are not for the fainthearted (p 18) is not consistent with the experience of others familiar with this technique. Likewise, oligonucleotide probes for screening recombinant phage (assuming the base sequences were known [page 19] would be an alternative to the plaque hybridization method described. As adequate or even excellent as chapters by MacLeod, Farrell, Talbot, Watson, and others may be, some chapters are wanting. Southern's

Detection of single base changes in a region of known sequence by colony probing of chromosomal DNA

Detection of single base changes in a region of known sequence by colony probing of chromosomal DNA