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Molecular biology
From Wikipedia, the free encyclopedia
Jump to: navigation, search

Molecular biology is the study of biology at a molecular level. The field overlaps
with other areas of biology and chemistry, particularly genetics and biochemistry.
Molecular biology chiefly concerns itself with understanding the interactions
between the various systems of a cell, including the interactions between DNA, RNA
and protein biosynthesis as well as learning how these interactions are regulated.
Writing in Nature in 1961, William Astbury described molecular biology as
not so much a technique as an approach, an approach from the viewpoint of the so-
called basic sciences with the leading idea of searching below the large-scale
manifestations of classical biology for the corresponding molecular plan. It is
concerned particularly with the forms of biological molecules and [...] is
predominantly three-dimensional and structural—which does not mean, however, that
it is merely a refinement of morphology. It must at the same time inquire into
genesis and function.[1]
•
[edit] Relationship to other "molecular-scale" biological sciences

Schematic relationship between biochemistry, genetics, and molecular biology

Researchers in molecular biology use specific techniques native to molecular
biology (see Techniques section later in article), but increasingly combine these
with techniques and ideas from genetics and biochemistry. There is not a defined
line between these disciplines. The following figure is a schematic that depicts
one possible view of the relationship between the fields:
• Biochemistry is the study of the chemical substances and vital processes
occurring in living organisms. Biochemists focus heavily on the role, function,
and structure of biomolecules. The study of the chemistry behind biological
processes and the synthesis of biologically active molecules are examples of
biochemistry.
• Genetics is the study of the effect of genetic differences on organisms.
Often this can be inferred by the absence of a normal component (e.g. one gene).
The study of "mutants" – organisms which lack one or more functional components
with respect to the so-called "wild type" or normal phenotype. Genetic
interactions (epistasis) can often confound simple interpretations of such "knock-
out" studies.
• Molecular biology is the study of molecular underpinnings of the process of
replication, transcription and translation of the genetic material. The central
dogma of molecular biology where genetic material is transcribed into RNA and then
translated into protein, despite being an oversimplified picture of molecular
biology, still provides a good starting point for understanding the field. This
picture, however, is undergoing revision in light of emerging novel roles for RNA.
Much of the work in molecular biology is quantitative, and recently much work has
been done at the interface of molecular biology and computer science in
bioinformatics and computational biology. As of the early 2000s, the study of gene
structure and function, molecular genetics, has been amongst the most prominent
sub-field of molecular biology.
Increasingly many other loops of biology focus on molecules, either directly
studying their interactions in their own right such as in cell biology and
developmental biology, or indirectly, where the techniques of molecular biology
are used to infer historical attributes of populations or species, as in fields in
evolutionary biology such as population genetics and phylogenetics. There is also
a long tradition of studying biomolecules "from the ground up" in biophysics.
[edit] Techniques of molecular biology
Since the late 1950s and early 1960s, molecular biologists have learned to
characterize, isolate, and manipulate the molecular components of cells and
organisms. These components include DNA, the repository of genetic information;
RNA, a close relative of DNA whose functions range from serving as a temporary
working copy of DNA to actual structural and enzymatic functions as well as a
functional and structural part of the translational apparatus; and proteins, the
major structural and enzymatic type of molecule in cells.
For more extensive list on protein methods, see protein methods.
For more extensive list on nucleic acid methods, see nucleic acid methods.
[edit] Expression cloning
Main article: Expression cloning
One of the most basic techniques of molecular biology to study protein function is
expression cloning. In this technique, DNA coding for a protein of interest is
cloned (using PCR and/or restriction enzymes) into a plasmid (known as an
expression vector). This plasmid may have special promoter elements to drive
production of the protein of interest, and may also have antibiotic resistance
markers to help follow the plasmid.
This plasmid can be inserted into either bacterial or animal cells. Introducing
DNA into bacterial cells can be done by transformation (via uptake of naked DNA),
conjugation (via cell-cell contact) or by transduction (via viral vector).
Introducing DNA into eukaryotic cells, such as animal cells, by physical or
chemical means is called transfection. Several different transfection techniques
are available, such as calcium phosphate transfection,electroporation,
microinjection and liposome transfection. DNA can also be introduced into
eukaryotic cells using viruses or bacteria as carriers, the latter is sometimes
called bactofection and in particular uses Agrobacterium tumefaciens. The plasmid
may be integrated into the genome, resulting in a stable transfection, or may
remain independent of the genome, called transient transfection.
In either case, DNA coding for a protein of interest is now inside a cell, and the
protein can now be expressed. A variety of systems, such as inducible promoters
and specific cell-signaling factors, are available to help express the protein of
interest at high levels. Large quantities of a protein can then be extracted from
the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity
under a variety of situations, the protein may be crystallized so its tertiary
structure can be studied, or, in the pharmaceutical industry, the activity of new
drugs against the protein can be studied.
[edit] Polymerase chain reaction (PCR)
Main article: Polymerase chain reaction
The polymerase chain reaction is an extremely versatile technique for copying DNA.
In brief, PCR allows a single DNA sequence to be copied (millions of times), or
altered in predetermined ways. For example, PCR can be used to introduce
restriction enzyme sites, or to mutate (change) particular bases of DNA, the
latter is a method referred to as "Quick change". PCR can also be used to
determine whether a particular DNA fragment is found in a cDNA library. PCR has
many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA,
and, more recently, real-time PCR (QPCR) which allow for quantitative measurement
of DNA or RNA molecules.
[edit] Gel electrophoresis
Main article: Gel electrophoresis
Gel electrophoresis is one of the principal tools of molecular biology. The basic
principle is that DNA, RNA, and proteins can all be separated by means of an
electric field. In agarose gel electrophoresis, DNA and RNA can be separated on
the basis of size by running the DNA through an agarose gel. Proteins can be
separated on the basis of size by using an SDS-PAGE gel, or on the basis of size
and their electric charge by using what is known as a 2D gel electrophoresis.
[edit] Macromolecule blotting and probing
The terms northern, western and eastern blotting are derived from what initially
was a molecular biology joke that played on the term Southern blotting, after the
technique described by Edwin Southern for the hybridisation of blotted DNA.
Patricia Thomas, developer of the RNA blot which then became known as the northern
blot actually didn't use the term[2]. Further combinations of these techniques
produced such terms as southwesterns (protein-DNA hybridizations), northwesterns
(to detect protein-RNA interactions) and farwesterns (protein-protein
interactions), all of which are presently found in the literature.
[edit] Southern blotting
Main article: Southern blot
Named after its inventor, biologist Edwin Southern, the Southern blot is a method
for probing for the presence of a specific DNA sequence within a DNA sample. DNA
samples before or after restriction enzyme digestion are separated by gel
electrophoresis and then transferred to a membrane by blotting via capillary
action. The membrane is then exposed to a labeled DNA probe that has a complement
base sequence to the sequence on the DNA of interest. Most original protocols used
radioactive labels, however non-radioactive alternatives are now available.
Southern blotting is less commonly used in laboratory science due to the capacity
of other techniques, such as PCR, to detect specific DNA sequences from DNA
samples. These blots are still used for some applications, however, such as
measuring transgene copy number in transgenic mice, or in the engineering of gene
knockout embryonic stem cell lines.
[edit] Northern blotting
Main article: northern blot
The northern blot is used to study the expression patterns of a specific type of
RNA molecule as relative comparison among a set of different samples of RNA. It is
essentially a combination of denaturing RNA gel electrophoresis, and a blot. In
this process RNA is separated based on size and is then transferred to a membrane
that is then probed with a labeled complement of a sequence of interest. The
results may be visualized through a variety of ways depending on the label used;
however, most result in the revelation of bands representing the sizes of the RNA
detected in sample. The intensity of these bands is related to the amount of the
target RNA in the samples analyzed. The procedure is commonly used to study when
and how much gene expression is occurring by measuring how much of that RNA is
present in different samples. It is one of the most basic tools for determining at
what time, and under what conditions, certain genes are expressed in living
tissues.
[edit] Western blotting
Main article: western blot
Antibodies to most proteins can be created by injecting small amounts of the
protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal
antibodies)or produced in cell culture (monoclonal antibodies). These antibodies
can be used for a variety of analytical and preparative techniques.
In western blotting, proteins are first separated by size, in a thin gel
sandwiched between two glass plates in a technique known as SDS-PAGE (sodium
dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are
then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This
membrane can then be probed with solutions of antibodies. Antibodies that
specifically bind to the protein of interest can then be visualized by a variety
of techniques, including colored products, chemiluminescence, or autoradiography.
Often, the antibodies are labeled with an enzymes. When a chemiluminescent
substrate is exposed to the enzyme it allows detection. Using western blotting
techniques allows not only detection but also quantitative analysis.
Analogous methods to western blotting can be used to directly stain specific
proteins in live cells or tissue sections. However, these immunostaining methods,
such as FISH, are used more often in cell biology research.
[edit] Eastern blotting
Main article: Eastern blotting
Eastern blotting technique is to detect post-translational modification of
proteins.[3] Proteins blotted on to the PVDF or nitrocellulose membrane are probed
for modifications using specific substrates.
[edit] Arrays
Main article: DNA microarray
A DNA array is a collection of spots attached to a solid support such as a
microscope slide where each spot contains one or more single-stranded DNA
oligonucleotide fragment. Arrays make it possible to put down a large quantity of
very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA
fragment molecule that is complementary to a single DNA sequence (similar to
Southern blotting). A variation of this technique allows the gene expression of an
organism at a particular stage in development to be qualified (expression
profiling). In this technique the RNA in a tissue is isolated and converted to
labeled cDNA. This cDNA is then hybridized to the fragments on the array and
visualization of the hybridization can be done. Since multiple arrays can be made
with the exact same position of fragments they are particularly useful for
comparing the gene expression of two different tissues, such as a healthy and
cancerous tissue. Also, one can measure what genes are expressed and how that
expression changes with time or with other factors. For instance, the common
baker's yeast, Saccharomyces cerevisiae, contains about 7000 genes; with a
microarray, one can measure qualitatively how each gene is expressed, and how that
expression changes, for example, with a change in temperature. There are many
different ways to fabricate microarrays; the most common are silicon chips,
microscope slides with spots of ~ 100 micrometre diameter, custom arrays, and
arrays with larger spots on porous membranes (macroarrays). There can be anywhere
from 100 spots to more than 10,000 on a given array.
Arrays can also be made with molecules other than DNA. For example, an antibody
array can be used to determine what proteins or bacteria are present in a blood
sample.
[edit] Allele Specific Oligonucleotide
Allele specific oligonucleotide (ASO) is a technique that allows detection of
single base mutations without the need for PCR or gel electrophoresis. Short (20-
25 nucleotides in length), labeled probes are exposed to the non-fragmented target
DNA. Hybridization occurs with high specificity due to the short length of the
probes and even a single base change will hinder hybridization. The target DNA is
then washed and the labeled probes that didn't hybridize are removed. The target
DNA is then analyzed for the presence of the probe via radioactivity or
fluorescence. In this experiment, as in most molecular biology techniques, a
control must be used to ensure successful experimentation. The Illumina
Methylation Assay is an example of a method that takes advantage of the ASO
technique to measure one base pair differences in sequence.
[edit] Antiquated technologies
In molecular biology, procedures and technologies are continually being developed
and older technologies abandoned. For example, before the advent of DNA gel
electrophoresis (agarose or polyacrylamide), the size of DNA molecules was
typically determined by rate sedimentation in sucrose gradients, a slow and labor-
intensive technique requiring expensive instrumentation; prior to sucrose
gradients, viscometry was used.
Aside from their historical interest, it is often worth knowing about older
technology, as it is occasionally useful to solve another new problem for which
the newer technique is inappropriate.
[edit] History
Main article: History of molecular biology
While molecular biology was established in the 1930s, the term was first coined by
Warren Weaver in 1938. Warren was the director of Natural Sciences for the
Rockefeller Foundation at the time and believed that biology was about to undergo
a period of significant change given recent advances in fields such as X-ray
crystallography. He therefore channeled significant amounts of (Rockefeller
Institute) money into biological fields.\

Reverse transcription polymerase chain reaction

From Wikipedia, the free encyclopedia
Jump to: navigation, search
"RT-PCR" redirects here. For real-time polymerase chain reaction, also called
quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase
chain reaction, see real-time polymerase chain reaction.
Reverse transcription polymerase chain reaction (RT-PCR) is a variant of
polymerase chain reaction (PCR), a laboratory technique commonly used in molecular
biology to generate many copies of a DNA sequence, a process termed
"amplification". In RT-PCR, however, RNA strand is first reverse transcribed into
its DNA complement (complementary DNA, or cDNA) using the enzyme reverse
transcriptase, and the resulting cDNA is amplified using traditional or real-time
PCR. Reverse transcription PCR is not to be confused with real-time polymerase
chain reaction (Q-PCR/qRT-PCR), which is also sometimes (incorrectly) abbreviated
as RT-PCR.
Contents
[hide]
• 1 RT-PCR principles and procedure
• 2 Use of reverse transcription polymerase chain reaction
• 3 See also
• 4 References
• 5 External links

[edit] RT-PCR principles and procedure

RT-PCR utilizes a pair of primers, which are complementary to a defined sequence
on each of the two strands of the cDNA. These primers are then extended by a DNA
polymerase and a copy of the strand is made after each cycle, leading to
logarithmic amplification [1].
RT-PCR includes three major steps. The first step is the reverse transcription
(RT) where RNA is reverse transcribed to cDNA using a reverse transcriptase and
primers. This step is very important in order to allow the performance of PCR
since DNA polymerase can act only on DNA templates [1]. The RT step can be
performed either in the same tube with PCR (one-step PCR) or in a separate one
(two-step PCR) using a temperature between 40°C and 50°C, depending on the
properties of the reverse transcriptase used [2].
The next step involves the denaturation of the dsDNA at 95°C, so that the two
strands separate and the primers can bind again at lower temperatures and begin a
new chain reaction. Then, the temperature is decreased until it reaches the
annealing temperature which can vary depending on the set of primers used, their
concentration, the probe and its concentration (if used), and the cations
concentration. The main consideration, of course, when choosing the optimal
annealing temperature is the melting temperature (Tm) of the primers and probes
(if used). The annealing temperature chosen for a PCR depends directly on length
and composition of the primers. This is the result of the difference of hydrogen
bonds between A-T (2 bonds) and G-C (3 bonds). An annealing temperature about 5
degrees below the lowest Tm of the pair of primers is usually used [3].
The final step of PCR amplification is the DNA extension from the primers which is
done by the thermostable Taq DNA polymerase usually at 72°C, which is the optimal
temperature for the polymerase to work. The length of the incubation at each
temperature, the temperature alterations and the number of cycles are controlled
by a programmable thermal cycler. The analysis of the PCR products depends on the
type of PCR applied. If a conventional PCR is used, the PCR product is detected
using agarose gel electrophoresis and ethidium bromide (or other nucleic acid
staining).
Conventional RT-PCR is a time-consuming technique with important limitations when
compared to real time PCR techniques [4]. This, combined with the fact that
ethidium bromide has low sensitivity, yields results that are not always reliable.
Moreover, there is an increased cross-contamination risk of the samples since
detection of the PCR product requires the post-amplification processing of the
samples. Furthermore, the specificity of the assay is mainly determined by the
primers, which can give false-positive results. However, the most important issue
concerning conventional RT-PCR is the fact that it is a semi or even a low
quantitative technique, where the amplicon can be visualised only after the
amplification ends.
Real time RT-PCR provides a method where the amplicons can be visualised as the
amplification progresses using a fluorescent reporter molecule. There are three
major kinds of fluorescent reporters used in real time RT-PCR, general non
specific DNA Binding Dyes such as SYBR Green I, TaqMan Probes and Molecular
Beacons (including Scorpions).
The real time PCR thermal cycler has a fluorescence detection threshold, below
which it cannot discriminate the difference between amplification generated signal
and background noise. On the other hand, the fluorescence increases as the
amplification progresses and the instrument performs data acquisition during the
annealing step of each cycle. The number of amplicons will reach the detection
baseline after a specific cycle, which depends on the initial concentration of the
target DNA sequence. The cycle at which the instrument can discriminate the
amplification generated fluorescence from the background noise is called the
threshold cycle (Ct). The higher the initial DNA concentration, the lower its Ct
will be.
[edit] Use of reverse transcription polymerase chain reaction
The exponential amplification via reverse transcription polymerase chain reaction
provides for a highly sensitive technique, where a very low copy number of RNA
molecules can be detected. Reverse transcription polymerase chain reaction is
widely used in the diagnosis of genetic diseases and, semiquantitatively, in the
determination of the abundance of specific different RNA molecules within a cell
or tissue as a measure of gene expression. Northern blot is used to study the
RNA's gene expression further. RT-PCR can also be very useful in the cloning of
eukaryotic genes in prokaryotes. Due to the fact that most eukaryotic genes
contain introns which are present in the genome but not in the mature mRNA, the
cDNA generated from a RT-PCR reaction is the exact (without regard to the error
prone nature of reverse transcriptases) DNA sequence which would be directly
translated into protein after transcription. When these genes are expressed in
prokaryotic cells for the sake of protein production/purification, the RNA
produced directly from transcription need not undergo splicing as the transcript
contains only exons (prokaryotes, such as E.coli, lack the mRNA splicing mechanism
of eukaryotes).
RT-PCR is commonly used in studying the genomes of viruses whose genomes are
composed of RNA, such as Influenzavirus A and retroviruses like HIV.

Real-time polymerase chain reaction

From Wikipedia, the free encyclopedia
(Redirected from Real time pcr)
Jump to: navigation, search
For reverse transcription polymerase chain reaction (RT-PCR), see reverse
transcription polymerase chain reaction.
In molecular biology, real-time polymerase chain reaction, also called
quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic
polymerase chain reaction, is a laboratory technique based on the PCR, which is
used to amplify and simultaneously quantify a targeted DNA molecule. It enables
both detection and quantification (as absolute number of copies or relative amount
when normalized to DNA input or additional normalizing genes) of one or more
specific sequences in a DNA sample.
The procedure follows the general principle of polymerase chain reaction; its key
feature is that the amplified DNA is detected as the reaction progresses in real
time, a new approach compared to standard PCR, where the product of the reaction
is detected at its end. Two common methods for detection of products in real-time
PCR are: (1) non-specific fluorescent dyes that intercalate with any double-
stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides
that are labelled with a fluorescent reporter which permits detection only after
hybridization of the probe with its complementary DNA target.
Frequently, real-time PCR is combined with reverse transcription to quantify
messenger RNA and Non-coding RNA in cells or tissues.
Abbreviations used for real-time PCR methods vary widely and include RTQ-PCR, Q-
PCR or qPCR. [1] Real-time reverse-transcription PCR is often denoted as qRT-PCR,
[2], RRT-PCR,[3] or RT-rt PCR.[4] The acronym RT-PCR commonly denotes reverse-
transcription PCR and not real-time PCR, but not all authors adhere to this
convention.[5]
Contents
[hide]
• 1 Background
• 2 Real-time PCR using double-stranded DNA dyes
• 3 Fluorescent reporter probe method
• 4 Quantification
• 5 Applications of real-time polymerase chain reaction
• 6 References
• 7 Further reading
• 8 External links

[edit] Background

Real time quantitative PCR uses fluorophores in order to detect levels of gene
expression.
Cells in all organisms regulate gene expression and turnover of gene transcripts
(messenger RNA, abbreviated to mRNA), and the number of copies of an mRNA
transcript of a gene in a cell or tissue is determined by the rates of its
expression and degradation.
Northern blotting is often used to estimate the expression level of a gene by
visualizing the abundance of its mRNA transcript in a sample. In this method,
purified RNA is separated by agarose gel electrophoresis, transferred to a solid
matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe
that is complementary to the gene of interest. Although this technique is still
used to assess gene expression, it requires relatively large amounts of RNA and
provides only qualitative or semiquantitative information of mRNA levels.
In order to robustly detect and quantify gene expression from small amounts of
RNA, amplification of the gene transcript is necessary. The polymerase chain
reaction is a common method for amplifying DNA; for mRNA-based PCR the RNA sample
is first reverse transcribed to cDNA with reverse transcriptase.
Development of PCR technologies based on reverse transcription and fluorophores
permits measurement of DNA amplification during PCR in real time, i.e., the
amplified product is measured at each PCR cycle. The data thus generated can be
analysed by computer software to calculate relative gene expression in several
samples, or mRNA copy number. Real-time PCR can also be applied to the detection
and quantification of DNA in samples to determine the presence and abundance of a
particular DNA sequence in these samples.
[edit] Real-time PCR using double-stranded DNA dyes
A DNA-binding dye binds to all double-stranded (ds)DNA in PCR, causing
fluorescence of the dye. An increase in DNA product during PCR therefore leads to
an increase in fluorescence intensity and is measured at each cycle, thus allowing
DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green will
bind to all dsDNA PCR products, including nonspecific PCR products (such as
"primer dimers"). This can potentially interfere with or prevent accurate
quantification of the intended target sequence.
1. The reaction is prepared as usual, with the addition of fluorescent dsDNA
dye.
2. The reaction is run in a thermocycler, and after each cycle, the levels of
fluorescence are measured with a detector; the dye only fluoresces when bound to
the dsDNA (i.e., the PCR product). With reference to a standard dilution, the
dsDNA concentration in the PCR can be determined.
Like other real-time PCR methods, the values obtained do not have absolute units
associated with it (i.e. mRNA copies/cell). As described above, a comparison of a
measured DNA/RNA sample to a standard dilution will only give a fraction or ratio
of the sample relative to the standard, allowing only relative comparisons between
different tissues or experimental conditions. To ensure accuracy in the
quantification, it is usually necessary to normalize expression of a target gene
to a stably expressed gene (see below). This can correct possible differences in
RNA quantity or quality across experimental samples.
[edit] Fluorescent reporter probe method
Fluorescent reporter probes detect only the DNA containing the probe sequence;
therefore, use of the reporter probe significantly increases specificity, and
enables quantification even in the presence of non-specific DNA amplification.
Fluorescent probes can be used in multiplex assays—for detection of several genes
in the same reaction—based on specific probes with different-coloured labels,
provided that all targeted genes are amplified with similar efficiency. The
specificity of fluorescent reporter probes also prevents interference of
measurements caused by primer dimers, which are undesirable potential by-products
in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect
of the primer dimers, which may depress accumulation of the desired products in
the reaction.
The method relies on a DNA-based probe with a fluorescent reporter at one end and
a quencher of fluorescence at the opposite end of the probe. The close proximity
of the reporter to the quencher prevents detection of its fluorescence; breakdown
of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the
reporter-quencher proximity and thus allows unquenched emission of fluorescence,
which can be detected after excitation with a laser. An increase in the product
targeted by the reporter probe at each PCR cycle therefore causes a proportional
increase in fluorescence due to the breakdown of the probe and release of the
reporter.
1. The PCR is prepared as usual (see PCR), and the reporter probe is added.
2. As the reaction commences, during the annealing stage of the PCR both probe
and primers anneal to the DNA target.
3. Polymerisation of a new DNA strand is initiated from the primers, and once
the polymerase reaches the probe, its 5'-3-exonuclease degrades the probe,
physically separating the fluorescent reporter from the quencher, resulting in an
increase in fluorescence.
4. Fluorescence is detected and measured in the real-time PCR thermocycler, and
its geometric increase corresponding to exponential increase of the product is
used to determine the threshold cycle (CT) in each reaction.

(1) In intact probes, reporter fluorescence is quenched. (2) Probes and the
complementary DNA strand are hybridized and reporter fluorescence is still
quenched. (3) During PCR, the probe is degraded by the Taq polymerase and the
fluorescent reporter released.
[edit] Quantification
Quantifying gene expression by traditional methods presents several problems.
Firstly, detection of mRNA on a Northern blot or PCR products on a gel or Southern
blot is time-consuming and does not allow precise quantification. Also, over the
20-40 cycles of a typical PCR, the amount of product reaches a plateau determined
more by the amount of primers in the reaction mix than by the input
template/sample.
Relative concentrations of DNA present during the exponential phase of the
reaction are determined by plotting fluorescence against cycle number on a
logarithmic scale (so an exponentially increasing quantity will give a straight
line). A threshold for detection of fluorescence above background is determined.
The cycle at which the fluorescence from a sample crosses the threshold is called
the cycle threshold, Ct. The quantity of DNA theoretically doubles every cycle
during the exponential phase and relative amounts of DNA can be calculated, e.g. a
sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template.
Since all sets of primers don't work equally well, one has to calculate the
reaction efficiency first. Thus, by using this as the base and the cycle
difference C(t) as the exponent, the precise difference in starting template can
be calculated (in previous example, if efficiency was 1.96, then the sample would
have 7.53 times more template).
Amounts of RNA or DNA are then determined by comparing the results to a standard
curve produced by real-time PCR of serial dilutions (e.g. undiluted, 1:4, 1:16,
1:64) of a known amount of RNA or DNA. As mentioned above, to accurately quantify
gene expression, the measured amount of RNA from the gene of interest is divided
by the amount of RNA from a housekeeping gene measured in the same sample to
normalize for possible variation in the amount and quality of RNA between
different samples. This normalization permits accurate comparison of expression of
the gene of interest between different samples, provided that the expression of
the reference (housekeeping) gene used in the normalization is very similar across
all the samples. Choosing a reference gene fulfilling this criterion is therefore
of high importance, and often challenging, because only very few genes show equal
levels of expression across a range of different conditions or tissues. [6][7]
[edit] Applications of real-time polymerase chain reaction
There are numerous applications for real-time polymerase chain reaction in the
laboratory. It is commonly used for both diagnostic and basic research.
Diagnostic real-time PCR is applied to rapidly detect nucleic acids that are
diagnostic of, for example, infectious diseases, cancer and genetic abnormalities.
The introduction of real-time PCR assays to the clinical microbiology laboratory
has significantly improved the diagnosis of infectious diseases, [8] and is
deployed as a tool to detect newly emerging diseases, such as flu, in diagnostic
tests.[9]
In research settings, real-time PCR is mainly used to provide quantitative
measurements of gene transcription. The technology may be used in determining how
the genetic expression of a particular gene changes over time, such as in the
response of tissue and cell cultures to an administration of a pharmacological
agent, progression of cell differentiation, or in response to changes in
environmental conditions.

Pyrosequencing
From Wikipedia, the free encyclopedia
Jump to: navigation, search

Example of a pyrogram showing the nucleotide sequence in a specific section of

DNA. The tops represent light emission and nucleotide binding.
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides
in DNA) based on the "sequencing by synthesis" principle. It differs from Sanger
sequencing, relying on the detection of pyrophosphate release on nucleotide
incorporation, rather than chain termination with dideoxynucleotides.[1] The
technique was developed by Pål Nyrén and his student Mostafa Ronaghi at the Royal
Institute of Technology in Stockholm in 1996.[2][3][4]
Contents
[hide]
• 1 Procedure
• 2 Commercialization
• 3 External links and references
• 4 References

[edit] Procedure
"Sequencing by synthesis" involves taking a single strand of the DNA to be
sequenced and then synthesizing its complementary strand enzymatically. The
Pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA
synthesizing enzyme) with another chemiluminescent enzyme. Essentially, the method
allows sequencing of a single strand of DNA by synthesizing the complementary
strand along it, one base pair at a time, and detecting which base was actually
added at each step. The template DNA is immobilized, and solutions of A, C, G, and
T nucleotides are added and removed after the reaction, sequentially. Light is
produced only when the nucleotide solution complements the first unpaired base of
the template. The sequence of solutions which produce chemiluminescent signals
allows the determination of the sequence of the template.
ssDNA template is hybridized to a sequencing primer and incubated with the enzymes
DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates
adenosine 5´ phosphosulfate (APS) and luciferin.
1. The addition of one of the four deoxynucleotide triphosphates (dNTPs)(in the
case of dATP we add dATPαS which is not a substrate for a luciferase) initiates
the second step. DNA polymerase incorporates the correct, complementary dNTPs onto
the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.
2. ATP sulfurylase quantitatively converts PPi to ATP in the presence of
adenosine 5´ phosphosulfate. This ATP acts as fuel to the luciferase-mediated
conversion of luciferin to oxyluciferin that generates visible light in amounts
that are proportional to the amount of ATP. The light produced in the luciferase-
catalyzed reaction is detected by a camera and analyzed in a program.
3. Unincorporated nucleotides and ATP are degraded by the apyrase, and the
reaction can restart with another nucleotide.
Currently, a limitation of the method is that the lengths of individual reads of
DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-
1000 obtainable with chain termination methods (e.g. Sanger sequencing). This can
make the process of genome assembly more difficult, particularly for sequence
containing a large amount of repetitive DNA. As of 2007, pyrosequencing is most
commonly used for resequencing or sequencing of genomes for which the sequence of
a close relative is already available.
The templates for pyrosequencing can be made both by solid phase template
preparation (Streptavidin coated magnetic beads) and enzymatic template
preparation (Apyrase+Exonuclease).
[edit] Commercialization
The company Pyrosequencing AB in Uppsala, Sweden commercialized machinery and
reagents for sequencing short stretches of DNA using the pyrosequencing technique.
Pyrosequencing AB was renamed to Biotage in 2003 which was acquired by Qiagen in
2008[5]. Pyrosequencing technology was further licensed to 454 Life Sciences. 454
developed an array-based pyrosequencing technology which has emerged as a platform
for large-scale DNA sequencing. Most notable are the applications for genome
sequencing and metagenomics. GS FLX, the latest pyrosequencing platform by 454
Life Sciences (now owned by Roche Diagnostics), can generate 400 million
nucleotide data in a 10 hour run with a single machine. Each run would cost about
5,000-7,000 USD, pushing de novo sequencing of mammalian genomes into the million
dollar range.

Variants of PCR
From Wikipedia, the free encyclopedia
Jump to: navigation, search
This page assumes familiarity with the terms and components used in the Polymerase
Chain Reaction (PCR) process.
The versatility of PCR has led to a large number of variants:
Contents
[hide]
• 1 Basic modifications
• 2 Pretreatments and extensions
• 3 Other modifications
• 4 Primer modifications
• 5 DNA Polymerases
• 6 Mechanism modifications
• 7 Isothermal amplification methods
• 8 Additional reading
• 9 References

[edit] Basic modifications

Often only a small modification needs to be made to the standard PCR protocol to
achieve a desired goal:
• Multiplex-PCR uses several pairs of primers annealing to different target
sequences. This permits the simultaneous analysis of multiple targets in a single
sample. For example, in testing for genetic mutations, six or more amplifications
might be combined. In the standard protocol for DNA Fingerprinting, the targets
assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent
Probe Amplification (or MLPA) permits multiple targets to be amplified using only
a single pair or primers, avoiding the resolution limitations of multiplex PCR.
Multiplex PCR has also been used for analysis of microsatellites and SNPs.[1]
Variations in VNTR lengths in 6 individuals.
• Variable Number of Tandem Repeats (VNTR) PCR targets areas of the genome
that exhibit length variation. The analysis of the genotypes of the sample 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 the target DNA. It is
used in some sequencing methods and hybridization probing, to generate one DNA
strand as product. Thermocycling is carried out as in PCR, but with a limiting
amount or leaving out one of the primers. When the limiting primer becomes
depleted, replication increases arithmetically through extension of the excess
primer[2]. A modification of this process, named L'inear-After-The-Exponential-PCR
(or LATE-PCR), uses a limiting primer with a higher Melting temperature (Tm) than
the excess primer to maintain reaction efficiency as the limiting primer
concentration decreases mid-reaction[3]. (Also see Overlap-extension PCR).
• Some modifications are needed to perform long PCR. The original Klenow-based
PCR process did not generate products that were larger than about 400 bp. Taq
polymerase can however amplify targets of up to several thousand bp long[4]. Since
then, modified protocols with Taq enzyme have allowed targets of over 50 kb to be
amplified[5].

Nested PCR
• Nested PCR is used to increase the specificity of DNA amplification. Two
sets of primers are used in two successive reactions. In the first PCR, one pair
of primers is used to generate DNA products, which may contain products amplified
from non-target areas. The products from the first PCR are then used as template
in a second PCR, using one ('hemi-nesting') or two different primers whose binding
sites are located (nested) within the first set, thus increasing specificity.
Nested PCR is often more successful in specifically amplifying long DNA products
than conventional PCR, but it requires more detailed knowledge of the sequence of
the target.
• Quantitative PCR (or Q-PCR) is used to measure the specific amount of target
DNA (or RNA) in a sample. By measuring amplification only within the phase of true
exponential increase, the amount of measured product more accurately reflects the
initial amount of target. Special thermal cyclers are used that monitor the amount
of product during the amplification. Quantitative Real-Time PCR (QRT-PCR) methods
use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes,
such as TaqMan, to measure the amount of amplified product as the amplification
progresses.
• Hot-start PCR is a technique performed manually by heating the reaction
components to the DNA melting temperature (e.g. 95°C) before adding the
polymerase. In this way, non-specific amplification at lower temperatures is
prevented [6]. Alternatively, specialized reagents inhibit the polymerase's
activity at ambient temperature, either by the binding of an antibody, or by the
presence of covalently bound inhibitors that only dissociate after a high-
temperature activation step. 'Hot-start/cold-finish PCR' is achieved with new
hybrid polymerases that are inactive at ambient temperature and are only activated
at elevated temperatures.
• In Touchdown PCR, the annealing temperature is gradually decreased in later
cycles. The annealing temperature in the early cycles is usually 3-5°C above the
standard Tm of the primers used, while in the later cycles it is a similar amount
below the Tm. The initial higher annealing temperature leads to greater
specificity for primer binding, while the lower temperatures permit more efficient
amplification at the end of the reaction[7].
• Assembly PCR (also known as Polymerase Cycling Assembly or PCA) is the
synthesis of long DNA structures by performing PCR on a pool of long
oligonucleotides with short overlapping segments, to assemble two or more pieces
of DNA into one piece. It involves an initial PCR with primers that have an
overlap and a second PCR using the products as the template that generates the
final full-length product. This technique may substitute for ligation-based
assembly.[8]
• In Colony PCR, bacterial colonies are screened directly by PCR, for example,
the screen for correct DNA vector constructs. Colonies are sampled with a sterile
toothpick and a small quantity of cells transferred into a PCR mix. To release the
DNA from the cells, the PCR is either started with an extended time at 95°C (when
standard polymerase is used), or with a shortened denaturation step at 100°C and
special chimeric DNA polymerase[9].
• The Digital polymerase chain reaction simultaneously amplifies thousands of
samples, each in a separate droplet within an emulsion.
[edit] Pretreatments and extensions
The basic PCR process can sometimes precede or follow another technique:
• RT-PCR (or Reverse Transcription PCR) is used to reverse-transcribe and
amplify RNA to cDNA. PCR is preceded by a reaction using reverse transcriptase, an
enzyme that converts RNA into cDNA. The two reactions may be combined in a tube,
with the initial heating step of PCR being used to inactivate the transcriptase.
[4] The Tth polymerase (described below) has RT activity, and can carry out the
entire reaction. RT-PCR is widely used in expression profiling, which detects the
expression of a gene. It can also be used to obtain sequence of an RNA transcript,
which may aid the determination of the transcription start and termination sites
(by RACE-PCR) and facilitate mapping of the location of exons and introns in a
gene sequence.
• Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' (or adaptors)
that are first ligated to fragments of the target DNA. PCR primers that anneal to
the linker sequences are then used to amplify the target fragments. This method is
deployed for DNA sequencing, genome walking, and DNA footprinting.[10] A related
technique is Amplified fragment length polymorphism, which generates diagnostic
fragments of a genome.
• Methylation-specific PCR (MSP) is used to identify patterns of DNA
methylation at cytosine-guanine (CpG) islands in genomic DNA.[11] Target DNA is
first treated with sodium bisulfite, which converts unmethylated cytosine bases to
uracil, which is complementary to adenosine in PCR primers. Two amplifications are
then carried out on the bisulfite-treated DNA: One primer set anneals to DNA with
cytosines (corresponding to methylated cytosine), and the other set anneals to DNA
with uracil (corresponding to unmethylated cytosine). MSP used in Q-PCR provides
quantitative information about the methylation state of a given CpG island.
[edit] Other modifications
Main article: PCR optimization
Adjustments of the components in PCR is commonly used for optimal performance:
• The divalent magnesium ion (Mg++) is required for PCR polymerase activity.
Lower concentrations Mg++ will increase replication fidelity, while higher
concentrations will introduce more mutations.[citation needed]
• Denaturants(such as DMSO) can increase amplification specificity by
destabilizing non-specific primer binding. Other chemicals, such as glycerol, are
stabilizers for the activity of the polymerase during amplification. Detergents
(such as Triton X-100) can prevent polymerase stick to itself or to the walls of
the reaction tube.
• DNA polymerases occasionally incorporate mismatch bases into the extending
strand. High-fidelity PCR employs enzymes with 3'-5' exonuclease activity that
decreases this rate of mis-incorporation. Examples of enzymes with proofreading
activity include Pfu; adjustments of the Mg++ and dNTP concentrations may help
maximize the number of products that exactly match the original target DNA.
[citation needed]
[edit] Primer modifications
Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich
source of modification:
• Normally PCR primers are chosen from an invariant part of the genome, and
might be used to amplify a polymorphic area between them. In Allele-specific PCR
the opposite is done. At least one of the primers is chosen from a polymorphic
area, with the mutations located at (or near) its 3'-end. Under stringent
conditions, a mismatched primer will not initiate replication, whereas a matched
primer will. The appearance of an amplification product therefore indicates the
genotype. (For more information, see SNP genotyping.)
• InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting
that uses primers selected from segments repeated throughout a genome to produce a
unique fingerprint of amplified product lengths[12]. The use of primers from a
commonly repeated segment is called Alu-PCR, and can help amplify sequences
adjacent (or between) these repeats.
• Primers can also be designed to be 'degenerate' - able to initiate
replication from a large number of target locations. Whole genome amplification
(or WGA) is a group of procedures that allow amplification to occur at many
locations in an unknown genome, and which may only be available in small
quantities. Other techniques use degenerate primers that are synthesized using
multiple nucleotides at particular positions (the polymerase 'chooses' the
correctly matched primers). Also, the primers can be synthesized with the
nucleoside analog inosine, which hybridizes to three of the four normal bases. A
similar technique can force PCR to perform Site-directed mutagenesis. (also see
Overlap extension polymerase chain reaction)
• Normally the primers used in PCR are designed to be fully complementary to
the target. However, the polymerase is tolerant to mis-matches away from the 3'
end. Tailed-primers include non-complementary sequences at their 5' ends. A common
procedure is the use of linker-primers, which ultimately place restriction sites
at the ends of the PCR products, facilitating their later insertion into cloning
vectors.
• An extension of the 'colony-PCR' method (above), is the use of vector
primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector,
and a single set of primers are designed for the areas of the vector flanking the
insertion site. Amplification occurs for whatever DNA has been inserted[4].
• PCR can easily be modified to produce a labeled product for subsequent use
as a hybridization probe. One or both primers might be used in PCR with a
radioactive or fluorescent label already attached, or labels might be added after
amplification. These labeling methods can be combined with 'asymmetric-PCR'
(above) to produce effective hybridization probes.
[edit] DNA Polymerases
There are several DNA polymerases that are used in PCR:
• The Klenow fragment, derived from the original DNA Polymerase I from E.
coli, was the first enzyme used in PCR. Because of its lack of stability at high
temperature, it needs be replenished during each cycle, and therefore is not
commonly used in PCR.
• The bacteriophage T4 DNA polymerase was also initially used in PCR. It has a
higher fidelity of replication than the Klenow fragment, but is also destroyed by
heat.

Taq polymerase (PDB).

• The DNA polymerase from Thermus aquaticus (or Taq), was the first
thermostable polymerase used in PCR[4], and is still the one most commonly used.
The enzyme can be isolated from its native source, or from its cloned gene
expressed in E. coli.
• The Stoffel fragment is made from a truncated gene for Taq polymerase and
expressed in E. coli. It is lacking 5'-3' exonuclease activity, and may be able to
amplify longer targets than the native enzyme.[citation needed]
• Faststart polymerase is a variant of Taq polymerase that requires strong
heat activation, thereby avoiding non-specific amplification due to polymerase
activity at low temperature (see hot-start PCR above).
• Pfu DNA polymerase, isolated from the archean Pyrococcus furiosus, has
proofreading activity, and a 5-fold decrease in the error rate of replication
compared to Taq.[13] Since errors increase as PCR progresses, Pfu is the preferred
polymerase when products are to be individually cloned for sequencing or
expression.
• Vent polymerase is an extremely thermostable DNA polymerase isolated from
Thermococcus litoralis.
• Tth polymerase is a thermostable polymerase from Thermus thermophilus. It
has reverse transcriptase activity in the presence of Mn2+ ions, allowing PCR
amplification from RNA targets.
[edit] Mechanism modifications
Sometimes even the basic mechanism of PCR can be modified:

Inverse PCR.
• Unlike normal PCR, Inverse PCR allows amplification and sequencing of DNA
that surrounds a known sequence. It involves initially subjecting the target DNA
to a series of restriction enzyme digestions, and then circularizing the resulting
fragments by self ligation. Primers are designed to be extended outward from the
known segment, resulting in amplification of the rest of the circle. This is
especially useful in identifying sequences to either side of various genomic
inserts[14].
• Similarly, Thermal Asymmetric InterLaced PCR (or TAIL-PCR) is used to
isolate unknown sequences flanking a known area of the genome. Within the known
sequence, TAIL-PCR uses a nested pair of primers with differing annealing
temperatures. A 'degenerate' primer is used to amplify in the other direction from
the unknown sequence[15].
[edit] Isothermal amplification methods
Some DNA amplification protocols have been developed that may be used
alternatively to PCR:
• Helicase-dependent amplification is similar to traditional PCR, but uses a
constant temperature rather than cycling through denaturation and
annealing/extension steps. DNA Helicase, an enzyme that unwinds DNA, is used in
place of thermal denaturation.[16]
• PAN-AC also uses isothermal conditions for amplification, and may be used to
analyze living cells.[17][18]
• Nicking Enzyme Amplification Reaction referred to as NEAR, is isothermal,
replicating DNA at a constant temperature using a polymerase and nicking enzyme.

Allele specific oligonucleotide

From Wikipedia, the free encyclopedia
Jump to: navigation, search
An Allele Specific Oligonucleotide (or ASO) is a short piece of synthetic DNA
complementary to the sequence of a variable target DNA. It acts as a probe for the
presence of the target in a Southern blot assay or, more commonly, in the simpler
Dot blot assay. It is a common tool used in genetic testing, forensics, and
Molecular Biology research.
An ASO is typically an oligonucleotide of 15-21 nucleotide bases in length. It is
designed (and used) in a way that makes it specific for only one version, or
allele, of the DNA being tested. The length of the ASO, which strand it is chosen
from, and the conditions by which it is bound to (and washed from) the target DNA
all play a role in its specificity. These probes can usually be designed to detect
a difference of as little as 1 base in the target's genetic sequence, a basic
ability in the assay of Single Nucleotide Polymorphisms (SNPs), important in
genotype analysis and the Human Genome Project. To be detected after it has bound
to its target, the ASO must be labeled with a radioactive, enzymatic, or
fluorescent tag. The Illumina Methylation Assay technology takes advantage of ASO
to detect one base pair difference (cytosine versus thymine) to measure
methylation at a specific CpG site.
Contents
[hide]
• 1 Example
• 2 Alternatives
• 3 History
• 4 References
• 5 External links

[edit] Example

Binding of the "S" ASO probe to "S" DNA (top) or "A" DNA (bottom).
The human disease Sickle Cell Anemia is caused by a genetic mutation in the codon
for the sixth amino acid of the blood protein beta-hemoglobin. The normal DNA
sequence G-A-G codes for the amino acid glutamate, while the mutation changes the
middle adenine to a thymine, leading to the sequence G-T-G (G-U-G in the mRNA).
This altered sequence substitutes a valine into the final protein, distorting its
structure.
To test for the presence of the mutation in a DNA sample, an ASO probe would be
synthesized to be complementary to the altered sequence[1], here labeled as "S".
As a control, another ASO would be synthesized for the normal sequence "A". Each
ASO is fully complementary to its target sequence (and will bind strongly), but
has a single mismatch against its non-target allele (leading to weaker
interaction). The first diagram shows how the "S" probe is fully complementary to
the "S" target (top), but is partially mismatched against the "A" target (bottom).

Schematic of dot-blots using the "A" or "S" ASO probes.

A segment of the beta-hemoglobin genes in the sample DNA(s) would be amplified by
PCR, and the resulting products applied to duplicate support membranes as Dot
blots. The sample's DNA strands are separated with alkali, and each ASO probe is
applied to a different blot. After hybridization, a washing protocol is used which
can discriminate between the fully-complementary and the mismatched hybrids. The
mismatched ASOs are washed off of the blots, while the matched ASOs (and their
labels) remain.
In the second diagram, six samples of amplified DNA have been applied to each of
the two blots. Detection of the ASO label that remains after washing allows a
direct reading of the genotype of the samples, each with two copies of the beta-
hemoglobin gene. Samples 1 and 4 only have the normal "A" allele, while samples 3
and 5 have both the "A" and "S" alleles (and are therefore heterozygous carriers
of this recessive mutation). Samples 2 and 6 have only the "S" allele, and would
be affected by the disease. The small amount of 'cross hybridization' shown is
typical, and is considered in the process of interpreting the final results.
[edit] Alternatives
ASO analysis is only one of the methods used to detect genetic polymorphisms.
Direct DNA sequencing is used to initially characterize the mutation, but is too
laborious for routine screening. An earlier method, Restriction Fragment Length
Polymorphism (RFLP) didn't need to know the sequence change beforehand, but
required that the mutation affect the cleavage site of a Restriction Enzyme. The
RFLP assay was briefly adapted to the use of oligonucleotide probes[2], but this
technique was quickly supplanted by ASO analysis of polymerase chain reaction
(PCR) amplified DNA. The PCR technique itself has been adapted to detect
polymorphisms, as Allele specific PCR. However, the simplicity and versatility of
the combined PCR/ASO method has led to its continued use, including with non-
radioactive labels, and in a "reverse dot blot" format where the ASO probes are
bound to the membrane and the amplified sample DNA is used for hybridization.
[edit] History
The use of synthetic oligonucleotides as specific probes for genetic sequence
variations was pioneered by R. Bruce Wallace, working at the City of Hope National
Medical Center in Duarte, California. In 1979 Wallace and his coworkers reported
the use of ASO probes to detect variations in a single-stranded bacterial
virus[3], and later applied the technique to cloned human genes. In 1983[4] and
1985[1] Wallace's lab reported the detection of the mutation for Sickle Cell
Anemia in samples of whole genomic DNA, although this application was hampered by
the small amount of label that could be carried by the ASO[1].
Fortunately PCR, a method to greatly amplify a specific segment of DNA, was also
reported in 1985[2]. In less than a year PCR had been paired with ASO analysis[5].
This combination solved the problem of ASO labeling, since the amount of target
DNA could be amplified over a million-fold. Also, the specificity of the PCR
process itself could be added to that of the ASO probes, greatly reducing the
problem of spurious binding of the ASO to non-target sequences. The combination
was specific enough that it could be used in a simple Dot blot, avoiding the
laborious and inefficient Southern blot method.