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A list of chemical analysis methods with acronyms. A. Atomic absorption spectroscopy (AAS) Atomic emission spectroscopy (AES) Atomic fluorescence spectroscopy (AFS) ...
The last 10-12 bases at the 3' end of a primer are sensitive to initiation of polymerase extension and general primer stability on the template binding site. The effect of a single mismatch at these last 10 bases at the 3' end of the primer depends on its position and local structure, reducing the primer binding, selectivity, and PCR efficiency.
Polymerase chain reaction itself is the process used to amplify DNA samples, via a temperature-mediated DNA polymerase.The products can be used for sequencing or analysis, and this process is a key part of many genetics research laboratories, along with uses in DNA fingerprinting for forensics and other human genetic cases.
Additionally, primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of hybridization to a similar sequence nearby. A commonly used method for selecting a primer site is BLAST search, whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the ...
For example, lower concentrations of magnesium or other cations may prevent non-specific primer interactions, thus enabling successful PCR. A "hot-start" polymerase enzyme whose activity is blocked unless it is heated to high temperature (e.g., 90–98˚C) during the denaturation step of the first cycle, is commonly used to prevent non-specific ...
For example, oligonucleotides with a hairpin structure cannot act efficiently as a primer. However, after heating the reaction mix to the annealing temperature the primer will undergo a conformation change allowing the primer to form a linear structure instead, which enables the primer to attach to the target segment and begin PCR.
Analysis of the genotypes in the samples usually involves sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as short tandem repeats (or STRs) is the basis for DNA fingerprinting databases such as CODIS. Asymmetric PCR preferentially amplifies one strand of a double-stranded DNA target.
Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple sequences at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform.