|
What are the odds we'll be able to amplify dinosaur DNA? See
Jurassic Park and PCR
Technologies term index Related glossaries include Combinatorial libraries
& synthesis, Labels, Signaling & Detection, Microarrays,
PCR is a key technology for, and an important tool for molecular
diagnostics
and Sequencing
Biology Gene definitions, DNA, Sequences,
DNA & beyond
Agglutination-PCR (ADAP):
an ultrasensitive solution-phase method for
detecting antibodies.[1] Antibodies
bind to and agglutinate synthetic antigen–DNA conjugates,
enabling ligation of the DNA strands and subsequent quantification by qPCR.
Like other Immuno-PCR (IPCR) detection methods[2][3] ADAP
combines the specificity of antibody-antigen recognition and the
sensitivity of PCR. ADAP detects zepto- to attomoles of antibodies in 2 μL
of sample with a dynamic range spanning 5–6 orders of magnitude. For
example, ADAP allows to detect anti-thyroglobulin autoantibodies from
human patient plasma with a 1000-fold increased sensitivity over an
FDA-approved radioimmunoassay. ADAP also allows to simultaneously detect
multiple antibodies in one experiment, much more than ELISA or radioimmunoassay.
Wikipedia accessed 2018 Nov 8
https://en.wikipedia.org/wiki/Agglutination-PCR
Amplified Fragment Length Polymorphism Analysis: The detection of RESTRICTION
FRAGMENT LENGTH POLYMORPHISMS by selective PCR amplification of restriction
fragments derived from genomic DNA followed by electrophoretic analysis of the
amplified restriction fragments. MeSH
2008
ASO probe (Allele Specific Oligo):
The sequence of the oligo is designed in such a way to allow/ inhibit hybridization
in the spot where the mutant (resistant) allele differs from the wild type
(susceptible) allele. [Schlwindlein]
absolute quantification:
To express true value, scientists (including
the author) have been using absolute quantification … it does not appear
to be a good fit for gene quantification, and thus the use of this
terminology should be discouraged … at this low level, a probability rather
than an absolute number defines the true copy number value for a given
sample. Francois. Ferré “Key issues” Gene Quantification Birkhauser
1998
Attempts to state the number of copies of a specific RNA per cell or
unit mass of tissue. Requires a number of extra conditions and treatments
that relative quantification does not. WM Freeman et al “Quantitative
RT-PCR: Pitfalls and Potential” Biotechniques 26: 112-125 Jan 1999 Related
term: relative quantification.
Agglutination-PCR (ADAP): an
ultrasensitive solution-phase method for detecting antibodies.[1] Antibodies
bind to and agglutinate synthetic antigen–DNA conjugates,
enabling ligation of the DNA strands and subsequent quantification by qPCR.
Like other Immuno-PCR (IPCR) detection methods[2][3] ADAP
combines the specificity of antibody-antigen recognition and the
sensitivity of PCR. ADAP detects zepto- to attomoles of antibodies in 2 μL
of sample with a dynamic range spanning 5–6 orders of magnitude. For
example, ADAP allows to detect anti-thyroglobulin autoantibodies from
human patient plasma with a 1000-fold increased sensitivity over an
FDA-approved radioimmunoassay. ADAP also allows to simultaneously detect
multiple antibodies in one experiment, much more than ELISA or radioimmunoassay.
Wikipedia accessed 2018 Nov 8
https://en.wikipedia.org/wiki/Agglutination-PCR
amplicon:
In molecular
biology, an amplicon is a piece of DNA or RNA that
is the source and/or product of amplification or replication events.
It can be formed artificially, using various methods including polymerase
chain reactions (PCR) or ligase
chain reactions (LCR), or naturally
through gene
duplication. In this
context, amplification refers to the production of one or more copies of a
genetic fragment or target sequence, specifically the amplicon. As it refers
to the product of an amplification reaction, amplicon is used interchangeably
with common laboratory terms, such as "PCR product." Wikipedia accessed 2018
Feb 26
https://en.wikipedia.org/wiki/Amplicon
amplification: Narrower terms: gene amplification,
kinetic PCR, kinetic RT- PCR, LCR, microamplification, NASBA, nested PCR, nucleic acid amplification,
protein amplification, RNA amplification, transcript mediated amplification, target amplification; signal amplification;
RNA amplified antisense RNA;
amplimer:
A piece of DNA formed
as the products of natural or artificial amplification events,
as in a polymerase
chain reaction. Wiktionary
https://en.wiktionary.org/wiki/amplimer
anneal:
to
be capable of combining with complementary nucleic acid by a process of heating and
cooling. Meriam Webster
https://www.merriam-webster.com/dictionary/anneal
branched DNA bDNA: Direct detection of target sequences by
hybridization with a branched DNA probe and target specific oligonucleotides.
Alkaline phosphatase- conjugated oligonucleotides, complementary to the branched
DNA complex, are detected using a chemiluminescent substrate. Whereas PCR
amplifies target sequences, the bDNA assay amplifies signal. Urdea, M.S. et
al. Clinical Chemistry 35, 1571, 1989 Promega
branched DNA signal amplification assay:
A molecular probe technique that utilizes branched DNA (bDNA) as a means to amplify the hybridization signal. One end of the bDNA molecule is designed to bind a specific target, while the other end of the bDNA molecule contains many branches of DNA that are designed to bind a probe used for signal detection.
MeSH, 2001
capture probe:
Phage or antibody probes that bind proteins in a sample
such that their relative expression levels can be detected.
Broader term: probe; Related term: reporter probe
competitive
hybridization:
Another critical feature of most microarrays is that different samples (often
two - control and sample) can be hybridized, competitively, to the same array.
This competitive hybridization is possible because fluorescent dyes with
different emission spectra can be used on different samples, allowing samples
tagged with the various dyes to be discriminated in imaging. Related term: competitive
PCR.
competitive PCR
cPCR:
During the last few years, many efforts have
been made to provide suitable controls to convert PCR to a quantitative
method ... One group [of methods] relies on external calibration ... among
[these] the competitive PCR methods (cPCR) are the most robust and reliable.
They are based on a co- amplification of the target DNA (sample) with a homologous
or heterologous DNA standard (competitor) which competes with the sample
template DNA for the same set of PCR primers. S Rupf, K. Eschrich,
"Quantification of bacteria by competitive polymerase chain reaction" American
Laboratory: 44- 46, July 2000 An internal control, close in composition to the target nucleic acid,
competes with the latter for reagents (such as common primers) in the same
reaction tube … represents the prototype for the so- called end- point quantitative
methods, in which quantification is based on the amount of amplified material (amplicon) obtained at the last amplification cycle. F.
Ferré "Key issues"
in Gene Quantification Birkhauser 1998 Related terms competitive
RT-PCR; competitive immunoassay Labels, signaling & detection
competitive RT-PCR:
Should be that - a competition between a
known amount of a template and an unknown target. This method avoids difficulties
created by differences in the efficiency of the PCR reaction itself with
different template/ primer sets. A competitive template binds the same primers
but has been altered in some way (small deletions, point mutation) to provide
a product that is distinguishable from the target itself. [Laura De Francesco,
"Taking the Measure of the Message" The Scientist 12[23]: 20, Nov. 23, 1998]
Compare non-competitive RT-PCR
digital PCR dPCR:
Remains an important technology to have in diagnostic labs, and as more
groups add and use this technology, novel applications continue to emerge.
dMIQE guidelines:
Minimum Information for Publication of Quantitative Digital PCR Experiments
guidelines. http://www.nist.gov/mml/bmd/genetics/upload/clinchem_2013_206375_full.pdf
DNA amplification: See gene amplification, PCR
DNA probes: Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure
DNA- DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and
DNA :RNA hybrid- specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections.
MeSH, 1989
end-point:
The traditional measurements of product … analyze
the reaction after it is completed can be accomplished through the use
of fluorescent intercalating dyes or through measurement of incorporated
radioactivity by autoradiography or phosphor imaging … Southern blots or
fluorescence detection are also used. A third type uses solid- state approaches
in which a bound enzyme produces fluorescence or luminescence. Finally,
several laboratories measure the production of amplification products following
resolution by HPLC or capillary electrophoresis. WM Freeman et al. "Quantitative
RT-PCR: Pitfalls and Potential" Biotechniques 26: 112-125 Jan 1999 Compare
real
time.
FISH Fluorescence In Situ Hybridization:
A
type of IN SITU HYBRIDIZATION in which target sequences are stained with
fluorescent dye so their location and size can be determined using fluorescence
microscopy. This staining is sufficiently distinct that the hybridization
signal can be seen both in metaphase spreads and in interphase nuclei. MeSH,
1993 Broader terms: hybridization, in situ hybridization ISH.
Narrower term: chromosome painting
Related term: Labels,
signaling & detection fluorescence
gene amplification: An increase in the number of copies of a
specific gene in an organism. This can lead to the production of a corresponding
protein at elevated levels. IUPAC Compendium
A selective increase in the number of copies of a gene coding for a
specific protein without a proportional increase in other genes. It occurs
naturally via the excision of a copy of the repeating sequence from the
chromosome and its extrachromosomal replication in a plasmid, or via the
production of an RNA transcript of the entire repeating sequence of ribosomal
RNA followed by the reverse transcription of the molecule to produce an
additional copy of the original DNA sequence. Laboratory techniques have
been introduced for inducing disproportional replication by unequal crossing
over, uptake of DNA from lysed cells, or generation of extrachromosomal
sequences from rolling circle replication. MeSH, 1980
Broader term: nucleic
acid amplification
Narrower term: PCR
Related terms branched DNA, LCR, NASBA, nested PCR,
OLA, PNA, target amplification Narrower terms: kinetic PCR, real- time PCR; Related
term: Assays & screening
quantitative assays
Gene Quantification,
Technical Univ. Munich,
Germany http://www.wzw.tum.de/gene-quantification/
heteroduplex analysis: A method of detecting gene mutation by mixing
PCR- amplified mutant and wild- type DNA followed by denaturation and
reannealing. The resultant products are resolved by gel electrophoresis, with single base substitutions detectable under optimal electrophoretic conditions and gel formulations. Large base pair mismatches may also be analyzed by using
electron microscopy to visualize heteroduplex regions.
MeSH, 1999
hot-start: PCR reaction in which a necessary component for polymerization (polymerase,
magnesium, nucleotides, etc.) is withheld from the reaction until all other
components achieve a temperature exceeding the annealing temperature of the
primers. This process minimizes amplification artifacts associated with
low temperature extension of misprimed oligonucleotides. C.R. Newton et. al.
Nucleic Acids Research 17: 2503, 1989. Promega
hybridization:
1. The formation of stable duplexes of
two DNA and/ or RNA (complementary) strands via Watson- Crick base pairing
used for locating or identifying nucleotide sequences and to establish
the effective transfer of nucleic acid material to a new host. 2. The formation
of a novel diploid organism either by sexual processes or by protoplast
fusion. IUPAC Biotech
A chemical reaction in which single-stranded DNA or RNA
molecules combine to form double-stranded complexes, including, for example, the
famous DNA double helix. The reaction obeys the usual base-pairing rules,
sometimes called Watson- Crick base pairing, in which adenine (A) binds to
thymine (T) (or uracil [U], in the case of RNA), and cytosine (C) binds to
guanine (G). Binding occurs through the formation of hydrogen bonds between the
paired bases, which are much weaker than the covalent bonds that bind the
elements of each strand.
Narrower terms: active hybridization, competitive hybridization, in
situ hybridization ISH, passive hybridization.
Related terms: anneal, stringency; Microarrays
blotting
hybridization stringency:
The percentage of nucleotides which must match on two unrelated
single- stranded nucleic acid molecules before they will base pair with each other to form a duplex, given a certain set of physical and chemical conditions. The hybridization stringency is used to determine when a hybridization
probe and a target nucleic acid will come together, and can be set by the researcher by varying the conditions. In general, if the percentage of matching nucleotides is lower than 70 percent, the two
single- stranded nucleic acid molecules are considered nonhomologous and any hybridization is considered
nonstringent. Life Sciences Dictionary Broader term: stringency
immuno-PCR:
Techniques that combine nucleic
acid amplification with an antibody-based assay can dramatically increase
the sensitivity of conventional immunoassays. This review summarizes the
methodology and applications of one such protein detection technique that
has been used for the past 23 years—the immuno-polymerase chain reaction
(usually referred to as immuno-PCR or IPCR). The key component of an
immuno-PCR is a DNA–antibody conjugate that serves as a bridge to link the
solid-phase immunoreaction with nucleic acid amplification. The efficiency
of immuno-PCR enables a 10- to 109-fold increase in detection sensitivity
compared with that of ELISA. Advancements in immuno-PCR have included
improvements of production of the DNA–antibody conjugate, assay formats,
and readout methods. As an ultrasensitive protein assay, immuno-PCR has a
broad range of applications in immunological research and clinical
diagnostics. Immuno-PCR: An ultrasensitive immunoassay for biomolecular
detection, Jinming
Lia
Lunan Wanga
Analytica Chimica Acta
Volume 910, 3
March 2016, Pages 12-24
https://www.sciencedirect.com/science/article/pii/S0003267016300162?via%3Dihub
in situ hybridization ISH:
A technique that localizes specific nucleic acid sequences within intact
chromosomes, eukaryotic cells, or bacterial cells through the use of specific
nucleic acid-labeled probes. MeSH, 1993
Using labeled (radioactive or fluorescent) nucleic acid probes
-
allows researchers to quantify levels of specific mRNAs within a cell. From the Latin "in place".
Narrower terms: FISH, primed in situ labeling; Related term
comparative genomic hybridization; Broader term: hybridization
Jurassic Park and PCR:
Although GenBank lacks dinosaur DNA, fragments of genomes past can
be found here. A practical limit of about 100,000 years currently applies
to the age of recoverable DNA samples. Beyond this limit, hydrolysis of
the phosphate backbone of the DNA and oxidative damage to the bases that
make up the DNA sequence become too great to allow for efficient PCR amplification.
This is why deposition of significant amounts of dinosaur sequence (age
> 65 million years) in GenBank is unlikely to occur in the near future.
However, many DNA sequences arising from extinct organisms and ancient
genomes [including from a Neanderthal, the late Neolithic "Iceman", Egyptian mummies,
woolly mammoths, a quagga, a moa and medieval French rabbits] are in
the database today, and the number is expected to grow as technology for
the extraction and amplification of aged DNA progresses. "DNA Sequences
from Times Past in GenBank" NCBI News, Spring 1999 http://www.ncbi.nlm.nih.gov/Web/Newsltr/Spring99/spring99.htm#DNA
Sequences
kinetic RT-PCR:
Direct use of amplification
kinetics to quantify RNA without the use of a standard. Started as an attempt
to avoid the long development times of standard construction and the problems
of designing, storing and accurately quantifying the standard itself. ... never
been widely used, recent advances in detection methods suggest that this
concept has the greatest potential for future quantitative RT-PCR
development. WM Freeman et al “Quantitative RT- PCR: Pitfalls and Potential”
Biotechniques 26: 112- 125 Jan 1999
LCR Ligase Chain Reaction:
A DNA amplification technique based upon
the ligation of OLIGONUCLEOTIDE PROBES. The probes are designed to exactly match
two adjacent sequences of a specific target DNA. The chain reaction is
repeated in three steps in the presence of excess probe: (1) heat
denaturation of double- .stranded DNA, (2) annealing of probes to target
DNA, and (3) joining of the probes by thermostable DNA ligase. After the
reaction is repeated for 20- 30 cycles the production of ligated probe is
measured. MeSH, 2001
miniaturization,
PCR: We will create a single, integrated platform where PCR primers are
synthesized in tandem, and PCR amplification is initiated by the release of
the PCR primers, and progress of the PCR reactions is monitored in Real-Time.
Furthermore, such reactions will be multiplexed in a high-throughput device
with sub-microliter reaction volumes, and will allow at least 1536 PCR
reactions to be performed and monitored in parallel. PCR Miniaturization,
Genome Technology Center, Stanford Univ School of Medicine, 2007 http://med.stanford.edu/sgtc/technology/pcr.html
molecular beacons:
single-stranded
oligonucleotide hybridization probes that form a stem-and-loop structure. The
loop contains a probe sequence that is complementary to a target sequence, and
the stem is formed by the annealing of complementary arm sequences that are
located on either side of the probe sequence. A fluorophore is covalently linked
to the end of one arm and a quencher is covalently linked to the end of the
other arm. Molecular beacons do not fluoresce when they are free in solution.
However, when they hybridize to a nucleic acid strand containing a target
sequence they undergo a conformational change that enables them to fluoresce
brightly Public Health
Research Institute New Jersey Medical School - Rutgers, The State University of
New Jersey.
http://www.molecular-beacons.org/MB_introduction.html
multiplex:
A sequencing approach that uses several pooled samples,
greatly increasing sequencing speed. DOE
In general, primer- extension technologies are amenable to high-
throughput applications and automation, yet only very low levels of multiplexing
are possible. Higher multiplexing can be accomplished by combining primer-
extension technology with microarray
technology.
Originally a math term meaning multiple, later a 19th century
telecommunications term, dating from the telegraph. Oxford English
Dictionary
NASBA Nucleic Acid
Sequence Based Amplification:
Use Self Sustained Sequence Based
Amplification MeSH entry term 2001
non-competitive RT-PCR:
The native
signal is unaltered by the standard. An increasing series of standard amounts
is co- amplified with equal amounts of total experimental RNA; however,
this occurs under conditions in which there is no competition for the components
in the PCR. The quantification is therefore estimated on a linear scaled
graph. The amount of standard signal is plotted against the native signal.
When the lines intersect, they reach the equivalence point, and quantification
is achieved. .. Generally some estimate of the amount of native signal must
be made before deciding on the standard amounts, because they are designed
to differ by only one log above and below the native. WM Freeman et al
“Quantitative RT- PCR: Pitfalls and Potential” Biotechniques 26: 112-125 Jan
1999 Compare competitive RT- PCR.
nucleic acid amplification:
Narrower terms: DNA amplification, gene amplification, PCR, RNA amplification.
nucleic acid amplification techniques:
Laboratory techniques that
involve the in-vitro synthesis of many copies of DNA or RNA from one original template
MeSH, 2001
nucleic acid probes:
Nucleic acid which complements a specific mRNA or DNA molecule, or fragment thereof; used for hybridization studies in order to identify microorganisms and for genetic studies.
MeSH, 1989
Nucleic Acid
Testing NAT:
Nucleic
Acid Testing (NAT) - FDA
Dec
28, 2017 - Nucleic
Acid Testing (NAT)
for. Human Immunodeficiency. Virus Type 1 (HIV-1) and. Hepatitis C Virus (HCV): Testing,
Product Disposition, and. Donor Deferral and Reentry. Guidance
for Industry.
for plasma testing
PCR and NGS-Based Molecular Diagnostics
March 14-15, 2019 San Francisco, CA
Program |
Advances
Techniques and Tools for Precision Medicine
Advances in molecular
diagnostics technologies have sparked innovation, expanded research
capabilities, and enhanced clinical diagnostics. Cambridge Healthtech
Institute’s 6th Annual PCR and NGS-Based Diagnostics symposium puts an
emphasis on the NGS and PCR technologies that drive precision medicine and
showcases how they are being used to alter clinical outcomes. This event
will provide a comprehensive look at integrating molecular diagnostics
solutions for biomarker discovery and development, point-of-care,
companion diagnostics, and infectious disease.
See also Polymerase Chain
Reaction
PNA Peptide Nucleic Acid:
DNA analogs containing neutral amide backbone linkages composed of aminoethyl glycine units instead of the usual phosphodiester linkage of deoxyribose groups. Peptide nucleic acids have high biological stability and higher affinity for complementary DNA or RNA sequences than analogous DNA
oligomers. MeSH, 1999
PNA probes (peptide nucleic acid)
:
Made of PNA rather than DNA, attempts to address the intrastrand hybridization
problem. A Marshall & J Hodgson “DNA chips: an array of possibilities”
Nature Biotechnology 16 (1): 27- 31 Jan 1998
Polymerase Chain
Reaction PCR: A laboratory technique to rapidly
amplify pre- determined regions of double- stranded DNA. Generally involves
the use of a heat stranded DNA polymerase. IUPAC Bioinorganic
In vitro method for producing large amounts of specific DNA or
RNA fragments of defined length and sequence from small amounts of short
oligonucleotide flanking sequences (primers). The essential steps include
thermal denaturation of the double- stranded target
molecules, annealing
of the primers to their complementary sequences, and extension of the annealed
primers by enzymatic synthesis with DNA polymerase. The reaction is efficient,
specific, and extremely sensitive. Uses for the reaction include disease
diagnosis, detection of difficult to isolate pathogens, mutation analysis,
genetic testing, DNA sequencing, and analyzing evolutionary relationships.
MeSH, 1991
Originally described in 1984 by Kary B. Mullis, who shared the Nobel Prize
for Chemistry for this invention in 1993, PCR enables the amplification of
specific nucleotide sequences through the use of a DNA polymerase. The sequence
to be amplified is identified through the use of synthetic oligonucleotides that
are complementary to the two terminal regions of the targeted sequence.
Broader
terms: gene amplification, nucleic acid
amplification and detection; Related terms: polymerase, primers, RT-PCR; Narrower term: Q-PCR
PCR and multiplex PCR: Guide and troubleshooting, Octavian Henegariu,
Yale Univ., US http://info.med.yale.edu/genetics/ward/tavi/PCR.html
Polymerase Chain Reaction (PCR) JumpStation, Horizon Press, UK http://www.highveld.com/pcr.html
Wikipedia http://en.wikipedia.org/wiki/PCR
polymerase DNA or RNA:
Any enzyme that catalyzes the formation of DNA or RNA from
deoxyribonucleotides or ribonucleotides. ORD Narrower term: Taq polymerase
primed in situ labeling:
A technique that labels specific sequences in whole chromosomes by in situ DNA chain elongation or PCR (polymerase chain reaction).
MeSH, 1999
primer- DNA:
Short sequences (generally about 10 base pairs) of DNA
that are complementary to sequences of messenger RNA and allow reverse
transcriptases to start copying the adjacent sequences of mRNA. Primers
are used extensively in genetic and molecular biology techniques. MeSH, 1994
A molecule that initiates the synthesis of a larger molecule. For example,
a short synthetic piece of DNA serves as a primer to initiate template- directed
DNA synthesis in PCR. NHLBI
Narrower term: primer extension.
Related term probes primer dimer: Artifacts, non- target
amplification products, caused by homologies within primers.
primer extension:
A method of SNP detection.
probes:
A specific DNA or RNA sequence which has been labelled
by radioactivity, fluorescence labels or chemiluminescence labels and which
is used to detect complementary sequences by hybridization techniques,
such as blotting or colony hybridization. IUPAC Compendium
Biomolecular probes used "to measure the presence or concentration of
biological molecules, biological structures, microorganisms, etc. by translating
a biochemical interaction at the probe surface into a quantifiable physical
signal. MeSH "biosensing techniques", 1999
Devices which use detector molecules to detect, investigate, or analyze
other molecules, macromolecules, molecular aggregates, or organisms. MeSH "molecular
probe techniques", 1991
Related terms:
oligonucleotide primers, target. Narrower terms: capture probes,
ASO probes, molecular beacons,
nucleic acid probes, PNA probes, padlock probes, RNA, probes;
Narrower [or equivalent?] terms: DNA probes, hybridization probes, Labels,
signal & detection See
also probes Microarrays
discussing the ambiguities of probe and target designations.
quantitative PCR: A
valuable technology used for diagnostics of diseases such as cancer and
infectious diseases, and for the detection of bacterial, fungal and viral
pathogens. The introduction of novel technology platforms such as digital PCR,
high-throughput platforms and improvements in automation and standardization
may provide useful tools for diagnosis, prognosis and therapeutic evaluations
for both the pharmaceutical industry and the medical community to move the
application of qPCR to the next level.
Despite recent attention focused on this technique,
quantitation is an old idea- almost as old as PCR itself. "Quantitative
PCR has been happening all along," says François Ferré, who
heads the gene quantification company Althea Technologies. In the early
1990s, for example, Michael Piatak, Ferré, and others used quantitative
PCR to show that HIV viral loads - the degree of infection - in patients'
blood were higher than previously thought (3, 4). "Even back in 1989, at
meetings focused on PCR, quantitation was a hot topic," Ferré says
.. As genes are located, their functions need to be determined, and studies
of gene expression become the focus. "The next dimension of research is
to figure out what is expressed and how much and when," says Mike Lucero,
product marketing manager for PCR at Perkin- Elmer. "That is just as basic
as knowing what the DNA sequence is." .. When many researchers say "quantitative
PCR", they mean kinetic or real-time PCR. Analytical Chemistry News & Features, March 1, 1999
191A-195A
Intended either to determine the number of copies of a given nucleic
acid sequence, or more generally to determine the relative abundance of
two sequences. J Peccoud and C Jacob "Statistical Estimations of PCR Amplification
Rates" in F. Ferré Gene Quantification Birkhauser 1998 Related terms: absolute quantification, gene
quantification, QRT- PCR, relative quantification,
relative QRT- PCR
quantitative RT-PCR QRT-PCR:
Has evolved into a widely used tool for
sensitive detection and quantitation of low- abundance RNA species. As the
focus of genomic research is shifting from the location of genes towards
functional genomics there is a growing demand for techniques capable of
accurately quantitating differences in mRNA levels in different settings. J. Stenman et al. Supplement to: Accurate determination of relative messenger
RNA levels by RT-PCR" Nature Biotechnology supp 17: 720- 722 July 1999
The RT step is the source of most of the variability in a quantitative
RT-PCR experiment. The final step in QRT- PCR is the detection and quantification
of amplification products. Inherently an indirect method of measurement.
WM Freeman et al "Quantitative RT-PCR: Pitfalls and Potential" Biotechniques
26: 112-125 Jan 1999 Related term: differential display Expression,
genes & beyond
Random Amplified Polymorphic DNA Technique RAPD: Technique that utilizes low stringency polymerase chain reaction
(PCR) amplification with single primers of arbitrary sequence to generate
strain specific arrays of anonymous DNA fragments. RAPD technique may be
used to determine taxonomic identity, assess kinship relationships, analyze
mixed genome samples, and create specific probes. MeSH, 1996
real time PCR:
[Russell] Higuchi and co- workers [at Roche] developed a system in which
PCR products can be detected in real time, meaning that the accumulation
of PCR products could be visualized at each cycle using an intercalating
dye, such as ethidium bromide, an ultraviolet source, and a CCD camera
… also referred to as "kinetic PCR". F. Ferre Gene Quantification
Birkhauser 1998
Real-time determinations monitor the reaction in the thermal cycler
as it progresses …offers the potential for improved quantification. The
errors in sample manipulation for end- point quantification are minimized
and a great deal more information about the PCR is obtained from the data
points for each cycle. WM Freeman et al "Quantitative RT- PCR: Pitfalls
and Potential" Biotechniques 26: 112-125 Jan 1999 Compare end-point Related terms:
kinetic PCR, kinetic RT-PCR
relative quantification:
The fact that relative quantification
is perceived as poor quantitative information has led to the false impression
that it is essential to publish copy numbers to increase the credibility
of the results. …methods capable of relative quantitation can provide extremely
valuable quantitative information. The quantitative power of such methods
will be directly proportional to the extent of their dynamic ranges and
to the tightness of their precision. F. Ferre “Key issues” in Gene
Quantification Birkhauser 1998
Determines the changes in steady-state
expression of a gene. For the purposes of the vast majority of
investigators, relative quantification is adequate. Semi- quantitative is
sometimes used as a synonym for relative quantification; however, it is
not an optimal term because of the confusion it causes and its imprecise
nature. WM Freeman et al “Quantitative RT-PCR: Pitfalls and
Potential” Biotechniques 26: 112-125 Jan 1999
Related
term: absolute quantification
relative quantitative RT-PCR: Uses an internal standard to monitor
each reaction and allow comparisons between different reactions to be made.
To do this, a second set of primers is incorporated into the reaction for
an invariant, housekeeping message. The difficulty here is matching the
level of the internal message to that of the target so that one reaction
doesn't dominate ... Finally, purely exogenous standards (template plus
primer) may be added to the reaction, which can give a signal against which
the unknown can be compared, providing a relative estimate of the amount of a
target species. Laura De Francesco, "Taking the Measure of the Message" The
Scientist 12[23]:20, Nov. 23, 1998
Reverse Transcriptase PCR: See
RT-PCR.
reverse transcriptases:
Enzymes found in retroviruses that can
synthesize complementary single strands of DNA from an mRNA sequence as
template. They are used in genetic engineering to produce specific cDNA
molecules from purified preparations of mRNA. IUPAC Compendium Related
term: RT-PCR
Rolling Circle Amplification RCA:
The derivative from of rolling circle replication has been successfully used
for amplification of DNA from
very small amounts of starting material.[1] This
amplification technique is named as Rolling circle amplification (RCA).
Different from conventional DNA amplification techniques such as polymerase
chain reaction (PCR),
RCA is an isothermal nucleic
acid amplification technique
where the polymerase continuously adds single nucleotides to a primer annealed
to a circular template which results in a long concatemer ssDNA that contains
tens to hundreds of tandem repeats (complementary to the circular template).[11]
Wikipedia accessed
2018 Feb 26
https://en.wikipedia.org/wiki/Rolling_circle_replication#Rolling_Circle_Amplification
Broader term: Rolling
circle replication
https://en.wikipedia.org/wiki/Rolling_circle_replication
RT-PCR Reverse Transcriptase Polymerase Chain Reaction:
A
variation of the PCR technique in which cDNA is made from RNA via reverse
transcription. The resultant cDNA is then amplified using standard PCR
protocols. MeSH, 1999
A method for assessing gene expression, detecting low copy number mRNA
transcripts, and generating complementary DNAs (cDNAs) for cloning. [Aileen
Constans "Lab Consumer Reverse Psychology" Scientist 14 (7): 29 Sept
4, 2000]
Narrower term: competitive RT- PCR
Related terms: Expression gene & protein
SBH Sequencing by Hybridization: A novel DNA sequencing technique in which an array (SBH chip) of short sequences of nucleotides
(probes) is brought in contact with a solution of (replicas of) the target DNA sequence. A biochemical method determines the subset of probes that bind to the target sequence (the spectrum of the sequence), and a combinatorial method is used to reconstruct the DNA sequence from the spectrum.
Franco Preparata, Eli Upfel, Samuel Health, Sequencing by Hybridization, Brown
Univ. 1999 http://www.cs.brown.edu/research/sbh/
self-sustained sequence replication:
An isothermal in-vitro nucleotide amplification process. The process involves the concomitant action of a
RNA- DIRECTED DNA POLYMERASE, a ribonuclease (RIBONUCLEASES), and a DNA-
DIRECTED RNA POLYMERASE to synthesize large quantities of
sequence- specific RNA and DNA molecules. MeSH, 2001
signal amplification methods:
Increasing the amount of signal per unit of
target for better detection. Because of the minute scale of hybridization, and the small amounts of target
present, it is desirable to increase the signal from an array. This signal-
boosting can be accomplished either by increasing the amount of target (target
amplification) or increasing the amount of signal per unit of target (signal
amplification). Narrower terms: branched DNA bDNA, Rolling Circle Amplification,
Hybrid
Capture® (HC) technology, Tyramide Signal Amplification TSA
stringency:
Reaction conditions - notably temperature, salt, and
pH - that dictate the annealing of single- stranded DNA/ DNA, DNA/ RNA, and
RNA/ RNA hybrids. At high stringency, duplexes form only between strands with perfect
one- to- one complementarity; lower stringency allows annealing between strands with some degree of mismatch between bases. Susan
A. Hagedorn [Life Sciences Dictionary] Narrower term: hybridization
stringency
Taq polymerase:
a thermostable DNA
polymerase named
after the thermophilic bacterium Thermus
aquaticus from
which it was originally isolated by Chien et al. in 1976.[1] Its
name is often abbreviated to Taq Pol or
simply Taq.
It is frequently used in the polymerase
chain reaction (PCR),
a method for greatly amplifying the quantity of short segments of DNA.
Wikipedia accessed 2018 Feb 26
https://en.wikipedia.org/wiki/Taq_polymerase
TaqMan probes
are hydrolysis probes that
are designed to increase the specificity of quantitative
PCR.
The method was first reported in 1991 by researchers at Cetus Corporation,[1] and
the technology was subsequently developed by Roche
Molecular Diagnostics for
diagnostic assays and by Applied
Biosystems (now
part of Thermo
Fisher Scientific)
for research applications. Wikipedia accessed 2018 Feb 26
https://en.wikipedia.org/wiki/TaqMan
target (hybridization), target amplification:
Drug targets
template: The nucleic acid single strand that is copied during
replication or transcription. IUPAC Biotech
whole genome amplification: The concept of whole genome amplification
is something that has arisen in the past few years as the polymerase chain
reaction (PCR) has been adapted to replicate regions of genomes that are of
biological interest. The applications are many - forensic science, embryonic
disease diagnosis, bioterrorism genome detection, "immortalization" of
clinical samples, microbial diversity, and genotyping. TL Hawkins et.
al. "Whole genome amplification: applications and advances" Current
Opinion in Biotechnology 13(1): 65- 67, Feb. 2002
PCR resources
PCR Glossary, Chang Bioscience 2002-2004
http://www.changbioscience.com/primo/pcr/index.htm
Real Time PCR Glossary, M Tevfik Dorak 2010 http://www.dorak.info/genetics/glosrt.html
How
to look for other unfamiliar terms
IUPAC definitions are reprinted with the
permission of the International Union of Pure and Applied Chemistry.
|
