Directed termination of the polymerase chain reaction: Kinetics and applications in mutation detection

Genome ◽  
1999 ◽  
Vol 42 (1) ◽  
pp. 72-79 ◽  
Author(s):  
Junjian Z Chen ◽  
Paul DN Hebert
Keyword(s):  
Polymerase Chain Reaction ◽  
Mutation Detection ◽  
Dna Amplification ◽  
Kinetic Mechanism ◽  
Single Step ◽  
Chain Termination ◽  
Chain Reaction ◽  
Nucleotide Substitutions ◽  
Polymerase Chain

We describe a PCR-based method (DT-PCR) that integrates both DNA amplification and directed chain termination into a single-step process. This method exploits unbalanced nucleotide concentrations to induce the polymerase chain reaction to terminate at specific nucleotide sites, leading to the generation of two sets of nested termination fragments from genomic DNA. The kinetic mechanism underlying the termination process is outlined and the application of this method to the detection and characterization of mutations in fragments as long as 1 kb is described. The method is effective for the analysis of both haploid and diploid genomes, and not only allows the recognition of both indels (insertions and deletions) and nucleotide substitutions, but also enables the determination of their position in a single-step fashion.Key words: DT-PCR, unbalanced dNTPs, chain termination, mutation detection.

DEOXYURIDINE TRIPHOSPHATES MODIFIED WITH AROMATIC GROUPS OF TYROSINE OR TRYPTOPHAN FOR DIRECT ELECTROCHEMICAL DETERMINATION OF DOUBLE-STRANDED DNA AMPLIFICATION PRODUCTS

2021 ◽  
Vol 1 (19) ◽  
pp. 248-250
Author(s):  
S.A. Khmeleva ◽  
G.R. Kutdusova ◽  
I.F. Duskaev ◽  
K.G. Ptitsyn ◽  
V.E. Kuznetsova ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Electrochemical Detection ◽  
Dna Amplification ◽  
Electrochemical Determination ◽  
Recombinase Polymerase Amplification ◽  
Chain Reaction ◽  
Double Stranded Dna ◽  
Polymerase Chain

A set of deoxyuridine triphosphates modified with aromatic groups of tyrosine or tryptophan was studied as substrates for amplification (by polymerase chain reaction or recombinase polymerase amplification) and as carriers of an electroactive ‘label’ for direct electrochemical detection of double-stranded DNA.

scholarly journals Using Polymerase Chain Reaction-based DNA Amplification to Fingerprint North American Potato Cultivars

HortScience ◽  
1996 ◽  
Vol 31 (1) ◽  
pp. 130-133 ◽  
Author(s):  
B. Sosinski ◽  
D.S. Douches
Keyword(s):  
Polymerase Chain Reaction ◽  
North American ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Chain Reaction ◽  
Arbitrary Primers ◽  
Polymerase Chain ◽  
Tuber Type

DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.

Improved methods for genotype determination of human alcohol dehydrogenase (ADH) at ADH 2 and ADH 3 loci by using polymerase chain reaction-directed mutagenesis

1990 ◽  
Vol 36 (10) ◽  
pp. 1765-1768 ◽  
Author(s):  
A Groppi ◽  
J Begueret ◽  
A Iron
Keyword(s):  
Polymerase Chain Reaction ◽  
Alcohol Dehydrogenase ◽  
Fragment Length Polymorphism ◽  
Dna Amplification ◽  
Directed Mutagenesis ◽  
Chain Reaction ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Improved Methods

Abstract The human gene for producing alcohol dehydrogenase (ADH; EC 1.1.1.1) is polymorphic at ADH 2 and ADH 3 loci. Until now, the study of this polymorphism required liver biopsy or allele-specific radioactive probes. We have used directed mutagenesis by the polymerase chain reaction (PCR) to amplify and analyze the genotype of ADH 2 and ADH 3 loci. Thus, we could determine easily and unambiguously the complete genotype at these two loci by using a microsample of blood and restriction fragment length polymorphism after DNA amplification by PCR.

PCR BY THERMAL CONVECTION

2004 ◽  
Vol 18 (16) ◽  
pp. 775-784 ◽  
Author(s):  
DIETER BRAUN
Keyword(s):  
Polymerase Chain Reaction ◽  
Thermal Convection ◽  
Dna Amplification ◽  
Reaction Vessel ◽  
Cold Region ◽  
Chain Reaction ◽  
Heating And Cooling ◽  
Highly Sensitive ◽  
Specific Amplification ◽  
Polymerase Chain

The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

Genetic diversity and phylogeny analysis of Azolla based on DNA amplification by arbitrary primers

Genome ◽  
1993 ◽  
Vol 36 (4) ◽  
pp. 686-693 ◽  
Author(s):  
Benoit Van Coppenolle ◽  
Iwao Watanabe ◽  
Charles Van Hove ◽  
Gerard Second ◽  
Ning Huang ◽  
...  
Keyword(s):  
Principal Component Analysis ◽  
Polymerase Chain Reaction ◽  
Principal Component ◽  
Dna Amplification ◽  
Component Analysis ◽  
Genetic Distances ◽  
Group Method ◽  
Chain Reaction ◽  
Arbitrary Primers ◽  
Polymerase Chain

The polymerase chain reaction was used to amplify random sequences of DNA from 25 accessions of Azolla to evaluate the usefulness of this technique for identification and phylogenetic analysis of this aquatic fern. Accessions were selected to represent all known species within the genus Azolla and to encompass the worldwide distribution of the fern. Primers of 10 nucleotides with 70% G + C content were used to generate randomly amplified polymorphic DNA from the symbiotic Azolla–Anabaena complex. Twenty-two primers were used and each primer gave 4–10 bands of different molecular weights for each accession. Bands were scored as present or absent for each accession and variation among accessions was quantified using Nei's genetic distances. A dendrogram summarizing phenetic relationships among the 25 accessions was generated using the unweighted pair-group method with arithmetic mean. Principal component analysis was also used to evaluate genetic similarities. Three distinct groups were identified: group 1 contains five species, group 2 contains the pinnata species, and group 3 contains the nilotica species. The analysis demonstrates that the major groups of Azolla species can be easily distinguished from one an other and, in addition, that closely related accessions within species can be identified. We further found that using 10 primers, a phylogeny that is essentially the same as that derived from 22 primers can be constructed. Our results suggest that total DNA extracted from the Azolla–Anabaena symbionts is useful for classification and phylogenetic studies of Azolla.Key words: Azolla–Anabaena symbiosis, genetic distances, polymerase chain reaction, principal component analysis.

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