PCR additives, including formamide, DMSO, glycerol, betaine, and PCRx
Enhancer Solution can enhance amplification. Their proposed mechanism is
to lower the melting temperature, thereby aiding primer annealing and
helping the DNA polymerase extend through regions of secondary structure.
Amplification of a DNA target
by the polymerase chain reaction (PCR) often requires laborious
optimization efforts. In this regard, the use of certain organic chemicals
such as dimethyl sulfoxide, polyethylene glycol, betaine and formamide as
cosolvents has been found to be very helpful. Unfortunately, very little
is known about the precise structural features that make these additives
effective and, accordingly, the number of such chemicals currently known
to enhance PCR is limited
The enhancement of PCR amplification by low
molecular weight amides
Raj Chakrabarti and Clarence E. Schutt, 2001
See more about
capture assay for nucleic acids that mimic enzyme linked immunosorbant
assays. In this assay, PCR products hybridized to an immobilized capture
PCR-ELISA is PCR that is followed
by capture and hybridization in microtiter plates to labeled probes and
detection similar to ELISA.
Plum Pox Potyvirus
Disease of Stone Fruits
Laurene Levy et al., 2000
PCR-RFLP (Polymerase Chain Reaction - Restriction
Alternative denomination: cleaved amplified
-Following PCR amplification
of a locus, the amplicon is treated with a restriction endonuclease. If
the recognition site for this enzyme is present in the amplicon, two or
more restriction fragments are generated. Thus sequence variation between
individuals at the recognition site(s) can be detected by electrophoresis.
chain reaction single-strand conformation polymorphism)
PCR-SSCP is a simple
procedure where denatured PCR products are electrophoresed through a
non-denaturing polyacrylamide gel. The single strands adopt primary
conformations that are dependent on their nucleotide sequence and this
determines the rate at which they migrate through the gel matrix. Each PCR
product with a different sequence therefore, will be theoretically
represented by two bands corresponding to the two strands of the amplified
Pfu DNA polymerase
Pfu DNA polymerase is an enzyme found in the
hyperthermophilic archaeon Pyrococcus furiosus, where it functions in vivo
to replicate the organism's DNA. In vitro, Pfu is used to quickly amplify
DNA in the Polymerase Chain Reaction, where the enzyme serves the central
function of copying a new strand of DNA during each extension step.The
main difference between Pfu and alternative enzymes is Pfu's superior
thermostability and 'proofreading' properties compared to other thermostable polymerases.
Pfu DNA polymerase , Wikipedia, The Free Encyclopedia
A mutation that can be mapped to one specific site within a locus. A small
mutation that consists of the replacement (transition or transversion);
addition; or deletion ( frameshift) of one or a few bases.
An alteration in DNA sequence caused by a
single nucleotide base change, insertion, or deletion
A change in a single base pair of DNA.
Separation of molecules through a polyacrylamide gel matrix in an electric
field. Separation may depend upon size and charge of the molecules.
Electrophoresis of nucleic acids and proteins through polyacrylamide gels.
enzyme which assembles nucleic acid residues into DNA or RNA polymers.
Polymerases work from the DNA complement of the sequence to be built. DNA
polymerases copy DNA to DNA to replicate the genome before mitosis, while
RNA polymerases copy DNA to RNA as the first step in gene transcription
Glossary of Biotechnology Terms
General term for enzymes that carry
out the synthesis of nucleic acid, using a pre-existing nucleic acid
template and appropriate nucleotides (viz. ribonucleotides for RNA and
deoxyribonucleotides for DNA).
A polymerase is a naturally
occurring enzyme, a biological macromolecule that catalyzes the formation
and repair of DNA (and RNA). The accurate replication of all living matter
depends on this activity
polymorphism is a DNA sequence variation that is common in
the population...The arbitrary cut-off point
between a mutation and a polymorphism is 1 per cent. That is, to be
classed as a polymorphism, the least common allele must have a frequency
of 1per cent or more in the population. If the frequency is lower that
this, the allele is regarded as a mutation. ...
Richard Twyman, 2003
Differences in DNA sequences that occur naturally in a population. Single
nucleotide substitutions, insertions and deletions of nucleotides and
repetitive sequences (microsatellites) are all examples of a polymorphism.
The position at which such a sequence difference is found is a polymorphic
PCR primers are short fragments of single stranded DNA (15-30 nucleotides
in length) that are complementary to DNA sequences that flank the target
region of interest. The purpose of PCR primers is to provide a “free”
3’-OH group to which the DNA polymerase can add dNTPs.
A primer is a short
segment of nucleotides which is complementary to a section of the DNA
which is to be amplified in the PCR reaction. Primers are annealed to the
denatured DNA template to provide an initiation site for the elongation of
the new DNA molecule
A short piece of DNA, usually synthetic, that defines the specific site on
a DNA molecule for a DNA polymerase to start making new DNA. An essential
ingredient in the PCR reaction mix.
See more about
Single-stranded DNA or RNA
molecules of specific base sequence, labeled either radioactively or
immunologically, that are used to detect the complementary base sequence
Defined nucleic acid (DNA or RNA) that can be used to identify, usually
through autoradiography, specific DNA or RNA molecules bearing the
complementary sequence. A labeled (radioactive; antigen; enzyme;
fluorescent) nucleic acid complementary to the sequence being searched for
in a restriction digest, genome library, northern blot or in situ
A finite nucleic acid piece that
can be used to identify specific DNA segments bearing its complementary
synthesis, the process of recognizing a basepair error during the
polymerization events and correcting it.
Fidelity of DNA
replication in vivo is achieved, in part, by the proof-reading activity of
the DNA polymerase. When an errant nucleotide is incorporated and forms a
mismatch with the template, it is removed by a 3´
to 5´ exonuclease proof-reading activity associated
with the polymerase. Some thermostable DNA polymerases have proof-reading
activity, a characteristic desirable for accurate DNA amplification and
for PCR amplification of long DNA sequences. The thermostable DNA
polymerase most widely used for PCR is Taq polymerase, which lacks
Detection of known mutation by
Wanli Bi and Peter J. Stambrook,
Laboratory procedures manual.