multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than

one pair of
primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction
tube have been described.The most useful of these are the empirical choice of oligonucleotide primers and the use of
hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to
facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex
PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids.)

Quantitative polymerase chain reaction

From Wikipedia, the free encyclopedia

(Redirected from QPCR)

Jump to: navigation, search

Quantitative polymerase chain reaction (qPCR) is a modification of the polymerase chain reaction used to rapidly
measure the quantity of DNA, complementary DNA or ribonucleic acid present in a sample. Like other forms of
polymerase chain reaction, the process is used to amplify DNA samples, via the temperature-mediated enzyme DNA
polymerase.

PCR theoretically amplifies DNA exponentially, doubling the number of molecules present with each amplification
cycle. The number of amplification cycles and the amount of PCR end-product should allow one to calculate the initial
quantity of genetic material, but numerous factors complicate this calculation. The ethidium bromide staining typically
used to assess a successful PCR prevents further amplification, and is only semi-quantitative. The polymerase chain
reaction may not be exponential for the first several cycles, and furthermore, plateaus eventually, so care must be taken
to measure the final amount of DNA while the reaction is still in the exponential growth phase. To overcome these
difficulties, several different quantitative methods have been developed.

The most sensitive quantification methods are done by the real-time polymerase chain reaction, where the amount of
DNA is measured after each cycle of PCR by use of fluorescent markers. Other end-point methods measure DNA after
PCR is completed. These methods depend on addition of a competitor RNA (for reverse-transcriptase PCR) or DNA in
serial dilutions or co-amplification of an internal control to ensure that the amplification is stopped while in the
exponential growth phase.

Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not to be confused
with reverse transcription polymerase chain reaction, which is also referred to as RT-PCR, but is used to amplify RNA
samples. The two methods may be used in concert to reverse transcribe RNA and then quantitate the resulting cDNA
using real-time PCR (often referred to as real-time RT-PCR).

Quantitative Real-Time Polymerase Chain Reaction for the Core Facility Using TaqMan and the
Perkin-Elmer/Applied Biosystems Division 7700 Sequence Detector

Deborah S. Grove

Nucleic Acid Facility, Life Science Consortium, The Pennsylvania State University, University Park, PA 16802

Quantitative real-time polymerase chain reaction (PCR) using the Perkin Elmer/Applied Biosystems Division 7700
Sequence Detector provides an accurate method for determination of levels of specific DNA and RNA sequences in
tissue samples. It is based on detection of a fluorescent signal produced proportionally during amplification of a PCR
product. Turn-around time for data acquisition and analysis by real-time PCR with the 7700 model is short, and results
are more reliable than by traditional PCR methods. This technology can be successfully offered as a service in a core
faculty setting. (J Biomol Tech 1999;10:11-16)

Key words: quantitative real-time polymerase chain reaction, Perkin Elmer/Applied Biosystems 7700 Sequence
Detector, nucleic acid sequence quantification.
Address correspondence and reprint requests to Deborah S. Grove, Nucleic Acid Facility, Life Science Consortium,
Pennsylvania State University, University Park, PA 16802.

Quantitative real-time polymerase chain reaction (PCR) using the Perkin Elmer/Applied Biosystems (PE/ABD) (Foster
City, CA, USA) Prism 7700 Sequence Detector System is a relatively new technology that provides a broad dynamic
range (at least five orders of magnitude) for detecting specific gene sequences with excellent sensitivity and precision.
DNA and RNA can be quantified using this detection system without laborious post-PCR processing.1,2 This
technology can be performed in a core facility environment because of the costs associated with instrument purchase,
setup, and maintenance.

Advantages

There are many advantages to quantifying gene sequences using this technology, foremost being sensitivity and
precision. This precision exists because quantitation of the gene sequence is determined by the Ct, which is calculated
during the exponential phase of the reaction. High specificity is conferred by the requirement of three oligos to anneal
to the DNA before any data are collected.

Turn-around time for data acquisition and analysis by real-time PCR with the 7700 is short. Setup and thermal cycling
require less than 3 to 4 hours and data analysis less than 10 additional minutes. Traditional PCR quantification may
require several days.

Competitive PCR is another technique often used to quantify DNA or RNA. Optimization of competitive PCR is
laborious and time consuming. Several dilutions of target sequences must be tested to achieve a suitable ratio of target
to competitor, and efficiencies of target and competitor must be similar. This assay is linear only over a very short
range compared with quantitation with the 7700. The number of samples that can be processed is also a limiting factor.

An advantage of providing the quantitative real-time PCR technology in a core facility setting is reduction of labor time
and costs. One half-time employee can easily process 96 samples per day, including setup and data analysis. The use of
robotics often found in core facilities allows preparation of samples with speed and accuracy.

Applications

The applications for quantitative real-time PCR are innumerable. Detection of genomic or viral DNA in tissues can be a
valuable diagnostic tool. Gene expression can be measured after extraction of total RNA and preparation of cDNA by a
reverse transcription (RT) step. Setup and analysis are simple and can more easily be extended to the clinical
environment than traditional PCR techniques.

One problem with developing applications is that most research conducted with this technology is done by
pharmaceutical companies. Because their work remains proprietary, successful TaqMan primers and probes, as well as
optimization conditions, are often not available. As a result, de novo design and optimization still require an investment
of time and money.

Examples of gene sequences that have been optimized for detection are shown in Table 1. My colleagues and I also
have optimized detection systems for prolactin, prolactin receptor, perforin, five isozymes of plant 1-
aminocyclopropane-1-carboxylic acid synthase (ACS), type X collagen, cartilage matrix protein, occludin, JC virus,
and CD19 (unpublished results).

A cDNA library is a collection of clones containing complementary DNA (cDNA) and is often intended to
represent the genes that are expressed within a given cell or tissue type at a given period.

Contents
[hide]
  1 Properties

 2 cDNA Library Construction

 3 cDNA Library uses

 3.1 cDNA Library vs. Genomic DNA Library

 4 External links

 5 References

 6 See also

[edit] Properties

 In some cases it is easier to isolate mRNA molecule than the gene itself, because of selective

expression of a particular gene, resulting in the relative abundance of that mRNA, for e.g., Insulin

mRNA in the islet cells of Pancreas, ovalbumin mRNA in the hen oviduct on treatment with estrogens.

In such cases, cDNA cloning is the approach of choice. Only about 1 to 10% of the total DNA in plant or

animal cell is expressed or is functional genetically. A cDNA library , which would thus represent only

the active genes, would be proportionately much smaller & hence much simpler to handle & construct.

Since the cDNA is derived from mRNA, there would be no intervening sequence in it & hence its

expression including translation in bacteria like E.coli will be straight forward Besides there are RNA

viruses, such as influenza virus & retroviruses which do not replicate via a DNA intermediate, only their

cDNA can be cloned.

[edit] cDNA Library Construction

Formation of a cDNA library.

cDNA is created from mRNA with the use of an enzyme known as reverse transcriptase. In eukaryotic cells,
a poly-(A) tail (consisting of a long sequence of adenine nucleotides) distinguishes mRNA from tRNA and
rRNA. Extracted RNA content from a cell can be passed through a chromatography column[1] | on which a
strand of complementary nucleotides7#151;such as short uracil [poly(U)] or thymidine extacts (oligo-dT) are
attached to a matrix in the column. The mRNA's poly-(A) tail can bind to the oligo-dTs[2], whilst the rest is
washed away. Elution with low-salt buffer releases the mRNA from the oligo-dTs.Once the purified mRNA is
recovered, reverse transcriptase is used to make templates of double-stranded DNA from the mRNA
templates. The newly formed DNA templates are then inserted into bacteria plasmids using restriction
enzymes (to cut open the plasmid) and ligase (to seal up the ends once the DNA has annealed into the
plasmid).Unfortunately, a very low percentage of plasmids actually have the DNA successfully inserted into
the plasmid despite a high percentage cut by restriction enzymes. Many plasmids simply remain cut open or
anneal with itself again. Therefore, to isolate those plasmids that do have the DNA successfully inserted, an
extra antibiotic resistance gene is usually inserted along with the DNA. The bacteria is then grown in a
medium with this antibiotic. All bacteria with plasmids that have successfully taken in the DNA (and hence
the antibiotic resistance), will then survive, with all the others dying off.The surviving colonies are then grown
to reproduce the plasmid with the DNA, which is then purified. This is also why these types of libraries are
called cDNA or complementary DNA, because it is a complemetary strand of DNA to the mRNA in question.

[edit] cDNA Library uses

Such a library has several uses. A cDNA of an eukaryotic organism (for example, a human) can be cloned
into a prokaryotic organism (for example, E. coli) and expressed (translated into the appropriate protein)
there (with limitations, for example posttranslational modification). A cDNA library is also important for
analysis through bioinformatics. The complete cDNA library of an organism gives the total of the proteins it
can possibly express. Also, comparison of cDNA sequence between libraries constructed from cells derived
from different organisms can provide insight into the genetic and evolutionary relationship between
organisms through the similarity of their cDNA.

[edit] cDNA Library vs. Genomic DNA Library

Why must cDNAs be used to create gene libraries from eukaryotic species?In Eukaryotic genomes, the
entire sequence of genes is often very large because coding sequence (exons) is interrupted by introns that
do not contain coding DNA sequence. The advantage of a cDNA library is that only the exons, or DNA
representing expressed sequence, are templates for the creation of DNA (via Reverse Transcriptase) to
collect the preferred genes. Prokarya generally do not contain the apparatus necessary to splice together
exons. Furthermore, the introns can increase the genomic space taken up by the gene to a point where
prokaryotic genetic manipulation schemes cannot handle the length of the DNA.When generating gene
libraries from prokaryotic species, Genomic DNA libraries are often used. Genes from prokaryotic cells
generally do not contain introns, and the DNA is therefore much shorter, and the RNA does not require
much post-processing to be expressed correctly. Also, prokaryotic mRNA can be difficult to isolate; because
of this a genomic library is more preferable for prokaryotic genomic studies.

Important Factors To Consider When Choosing Primers:

• Optimal Tm (annealing T) ~ 50-60°C

• Oligonucleotide GC-content should be between 40-60%.
• Calculated Tm (melting temperature) for both primers used in reaction should not differ >5°C.
• Primer annealing temperature is usually 5°C below the calculated lower Tm. However it should be chosen
empirically for individual conditions.
• Inner self-complementary hairpins of >4 and of dimers >8 should be avoided.
• 3' terminus is extremely case sensitive - it must not be complementary to any region of the other primer used
in the reaction and must provide correct base matching to template.
• If possible, primers should start and end with 1-2 G or C residues
• Primers should NOT have any extendisve secondary structure or self-complementarity (can result in Primer-
dimers).

The High Sensitivity of PCR

Due to the extremely high sensitivity of PCR, contamination from non-template PCR present in the lab
environment (from bacteria, viruses, and your own DNA) presents a real problem. Precautions therefore must be taken.
Several steps can be taken to prevent and minimize PCR contamination including the use of aerosol barrier tip filters,
laminar flow cabinet, and a special preparation area for PCR separate from the DNA isolation area.

Polymerase Errors

DNA polymerase are not perfect by any means, however they are quite good at what they do. Mistakes and
errors do happen in PCR by DNA polymerases in the test tube and in the human body. The polymerases used in PCR
often lack 3' to 5' exonuclease activity such as Taq polymerase. This enzyme lacks the ability to correct
misincorporated nucleotides, an activity which is present in higher eukaryotes.

A polymerase lacking the 3' to 5' exonuclease activity has a higher error rate, around 1 in 10000 bases are
misincorporated.

Recombinant polymerases have been generated and other polymerases have been isolated which are available to more
accurate PCR and have a variety of applications such as sequencing.

Examples of polymerases with 3' to 5' exonuclease activity include: Vent, which is extracted from Thermococcus
litoralis , Pfu which is extracted from Pyrococcus furiosus, and Pwo which is extracted from Pyrococcus woesii .

PCR Polymerase Enzyme Proficiency and PCR Product Size / Length Limitations

DNA polymerase are proficient enough to efficiently amplify DNA products up to a few thousand basepairs (2-5 kb).
PCRs of longer products are less efficient due to enzyme activity loss, and inaccuracies introduced by longer PCRs.
Adding fresh DNA polymerase helps with enzyme activity lost due to the half life of the polymerase, however this does
not help when accurate PCR is required.

It is possible to amplify PCR products up to 20kb using lower or slower heating cycles and special mixtures of
polymerases. Long and Accurate PCR is accomplished by using mixtures of processive polymerases and high fidelity
Polymerases.

Factors Affecting PCR:

PCR Reaction Volume

If you are increasing the PCR reaction volume proportionally to a PCR reaction that is optimized or works well, in
general you will increase the amount of product with increasing volume. Exceptions to this include older mineral oil
PCR machines and PCR thermocyclers without a heated-lid. In these cases, increasing the PCR reaction volume will
negatively affect the PCR product and efficiency of the reaction.

PCR Primer Design

Primer are usually chosen to be between 18-25 nucleotides in length. Longer primers also work 30-40 nucleotides or bp
during pcr

A GC clamp at the 5' ends of the primers helps.

One can thus design primers with 1 or 2 nucleotides GC at the start and ends of the pri Annealing
Temperature and Primer Design

Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the
melting temperature of a NA duplex increases both with its length, and with increasing (G+C) content: a simple
formula for calculation of the Tm is

Tm = 4(G + C) + 2(A + T)oC.

Thus, the annealing temperature chosen for a PCR depends directly on length and composition of the primer(s). One
should aim at using an annealing temperature (Ta) about 5oC below the lowest Tm of ther pair of primers to be used
(Innis and Gelfand, 1990). A more rigorous treatment of Ta is given by Rychlik et al. (1990): they maintain that if the
Ta is increased by 1oC every other cycle, specificity of amplification and yield of products <1kb in length are
both increased. One consequence of having too low a Ta is that one or both primers will anneal to sequences other
than the true target, as internal single-base mismatches or partial annealing may be tolerated: this is fine if one
wishes to amplify similar or related targets; however, it can lead to "non-specific" amplification and consequent
reduction in yield of the desired product, if the 3'-most base is paired with a target.

A consequence of too high a Ta is that too little product will be made, as the likelihood of primer annealing is
reduced; another and important consideration is that a pair of primers with very different Tas may never give
appreciable yields of a unique product, and may also result in inadvertent "asymmetric" or single-strand
amplification of the most efficiently primed product strand.

Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the
Tm, or unless they are unusually long.

Primer Length

The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner if one runs the risk of
having problems such as described above. Apart from the Tm, a prime consideration is that the primers should be
complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. (See
hybridn.doc).

For example, there is a ¼ chance (4-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-
2
) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen
base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about
the size of the human or maize genome, and 1000x greater than the genome size of E. coli. Thus, the association of a
greater-than-17-base oligonucleotide with its target sequence is an extremely sequence-specific process, far more so
than the specificity of monoclonal antibodies in binding to specific antigenic determinants. Consequently, 17-mer or
longer primers are routinely used for amplification from genomic DNA of animals and plants. Too long a primer
length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and non-specific
priming.

Degenerate Primers

For amplification of cognate sequences from different organisms, or for "evolutionary PCR", one may increase the
chances of getting product by designing "degenerate" primers: these would in fact be a set of primers which have a
number of options at several positions in the sequence so as to allow annealing to and amplification of a variety
of related sequences. For example, Compton (1990) describes using 14-mer primer sets with 4 and 5 degeneracies as
forward and reverse primers, respectively, for the amplification of glycoprotein B (gB) from related herpesviruses. The
reverse primer sequence was as follows:

TCGAATTCNCCYAAYTGNCCNT
 where Y = T + C, and N = A + G + C + T, and the 8-base 5'-terminal extension comprises a EcoRI site (underlined)
and flanking spacer to ensure the restriction enzyme can cut the product (the New England Biolabs catalogue gives a
good list of which enzymes require how long a flanking sequence in order to cut stub ends). Degeneracies obviously
reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases;
also, increased degeneracy means concentration of the individual primers decreases; thus, greater than 512-fold
degeneracy should be avoided. However, I have used primers with as high as 256- and 1024-fold degeneracy for the
successful amplification and subsequent direct sequencing of a wide range of Mastreviruses against a background of
maize genomic DNA (Rybicki and Hughes, 1990). Primer sequences were derived from multiple sequence alignments;
the mismatch positions were used as 4-base degeneracies for the primers (shown as stars; 5 in F and 4 in R), as shown
above. Despite their degeneracy, the primers could be used to amplify a 250 bp sequence from viruses differing in
sequence by as much as 50% over the target sequence, and 60% overall. They could also be used to very sensitively
detect the presence of Maize streak virus DNA against a background of maize genomic DNA, at dilutions as low as
1/109 infected sap / healthy sap. Some groups use deoxyinosine (dI) at degenerate positions rather than use
mixed oligos: this base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo
where it is present. This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture,
but may result in too high a degeneracy where there are 4 or more dIs in an oligo.

Choosing/designing PCR primers

In designing primers for PCR, the following steps/rules were tested and proven to be useful:

• length of individual primers between 18-24 bases. Longer primers (30-35 bp) seem to work in more similar
cycling conditions compared with shorter primers, and can make multiplexing easier (see picuters below).
• it is desirable (but not absolutely necessary) that the two primers have a close melting temperature or Tm (say,
within 5o C or so). If Tm difference between the two primers is high, the lower Tm can be increased by increasing
the length of that primer at the 3' end (this can also keep the size of the amplified locus constant) or the 5' end.
• purine:pyrimidine content around 1:1 (maybe 40-60%)
• if possible, primer sequence should start and end with 1-2 GC pairs
• each primer pair should be tested for primer-primer interactions. For this purpose a useful Macintosh program
is "CPrimer", a freeware available at ftp.bio.indiana.edu. This program also provides the melting temperature for
the sequences entered, thus helping in designing PCR programs. Very convenient, some web sites offer programs
that can be used directly on those sites to do the same functions: (search for optimal primers, melting
temperatures).
• primer sequences should be aligned with all DNA sequences entered in the databases (using BLAST programs)
and checked for similarities with repetitive sequences or with other loci, elwhere in the genome. If two loci are
very similar (for example across species) it is useful to design the primers so that at least 1-2 bases at the 3' end
are specific for the locus to be amplified

• cycling conditions and buffer concentrations should be adjusted for each primer pair, so that amplification of the
desired locus is specific, with no secondary products (see other pages). If this is not possible, the sequences of the
primers should be either elongated with 4-5 bases or simply, changed entirely.

Cycle Number

The number of amplification cycles necessary to produce a band visible on a gel depends largely on the starting
concentration of the target DNA: Innis and Gelfand (1990) recommend from 40 - 45 cycles to amplify 50 target
molecules, and 25 - 30 to amplify 3x105 molecules to the same concentration. This non-proportionality is due to a so-
called plateau effect, which is the attenuation in the exponential rate of product accumulation in late stages of a PCR,
when product reaches 0.3 - 1.0 nM. This may be caused by degradation of reactants (dNTPs, enzyme); reactant
depletion (primers, dNTPs - former a problem with short products, latter for long products); end-product inhibition
(pyrophosphate formation); competition for reactants by non-specific products; competition for primer binding by re-

annealing of concentrated (10nM) product (Innis and Gelfand, 1990).

AFLP PCR Background

Amplified Fragment Length Polymorphism PCR, also called AFLP PCR was originally described by Zabeau et al., 1993.

AFLP is composed of 3 steps:

1-A) Cellular DNA is digested with one or more restriction enzymes. Typically this involves a combination of two restriction
enzymes: a 4 base cutter (MseI) and a 6 base cutter (EcoRI).

1-B) Ligation of linkers (restriction half-site specific adaptors) to all restriction fragments.

2-A) Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences.

3) Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern. The aim of this tool is to
perform a theoretical AFLP-PCR experiment by using the same principles, and to suggest the adaptors and primers needed in the
experiment.

Applications of AFLP PCR

AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping
individuals for a large number of loci using a minimal number of PCR reactions.

What is Colony PCR?

The definition of Colony PCR is:

Colony PCR the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Selected colonies of bacteria
or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix
or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of
interest.

Inverse PCR Background

Inverse PCR also called IPCR, and was first described by Ochman et al. in 1988 (1).

A limitation of standard PCR is that 5' and 3' flanking regions of your DNA fragment of interest must be known. Inverse PCR allows
you to conduct PCR when you only have the information of one internal sequence.
Inverse polymerase chain reaction is a variant of PCR, and is used when only one internal sequence of the target DNA is
known. It is therefore very useful in identifying flanking DNA sequences of genomic inserts. Similar to other PCR methods, inverse
PCR amplifies target DNA using DNA polymerase.

Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usual
orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle.

The Inverse PCR Method The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a
restriction endonuclease. This cut results in a known sequence at either end of unknown sequences.

Inverse PCR Steps

1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion.
2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular
DNA ligation product.
3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known
internal sequence generating a linear product with known terminal sequences. This can now be used for PCR
(polymerase chain reaction).
4) Standard PCR is conducted with primers complementary to the now known internal sequences.

In summary: Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested
and then ligated to generate circular DNA. PCR primers pointing away from the known sequences are then
employed to amplify the flanking sequences.

Applications of Inverse PCR Inverse PCR has numerous applications in molecular biology including the amplification and
identification of sequences flanking transposable elements, and the identification of genomic inserts.

Reverse Transcription Polymerase Chain Reaction Reverse transcription polymerase chain reaction (RT-PCR) is based on the
polymerase chain reaction (PCR). More importantly it is based on the process of reverse transcription, which reverse transcribes RNA
into DNA and was initially isolated from retroviruses. The techniques of RT-PCR allows the formation of cDNA
(complementary or copy DNA) from RNA, which stores the sequence of RNA (such as messenger RNA, mRNA) in the more stable
form of nucleic acid, DNA. This reverse transcription from RNA into its reverse complement DNA (cDNA) is the first step of a
usually two-step process of RT-PCR. Furthermore, by copying the RNA into DNA, one can then amplify the cDNA sequence by
using primers specific for the DNA sequence. This amplification is the final second major step of the two-step process of RT-PCR.

Applications of RT-PCR .The exponential amplification of complementary sequence of mRNA or RNA

sequences via reverse transcription polymerase chain reaction allow for a high sensitivity detection
technique, where low copy number or less abundant RNA molecules can be detected. It is also used to
clone mRNA sequences in the form of complementary DNA, allowing libraries of cDNA (cDNA libraries) to
be created which contain all the mRNA sequences of genes expressed in a cell. Furthermore, it allows the
creation of cDNA constructs which were cloned by RT-PCR and allow the expression of genes at the RNA
and protein levels for further study.

Assembly PCR Protocol . Assembly PCR is a polymerase chain reaction variation that artificial synthesizes long DNA
sequences by performing PCR on a pool of long oligonucleotides (primers) with short overlapping segments. The oligonucleotides
alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby
selectively producing the final long DNA product.

Asymmetric PCR Protocol. Asymmetric PCR is used to preferentially amplify one strand of the target DNA
more than the other.

Applications of Asymmetric PCR

The Asymmetric PCR is useful in some sequencing and hybridization probing applications where having only one of the two
complementary stands is sufficient or required.

Asymmetric PCR Method

The asymmetric PCR method is conducted as the standard PCR protocol, however a great excess of the primers for the chosen strand
is used. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR
are required.

.Primers for Asymmetric PCR

A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations. As asymmetric
PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration
primer continues to primer synthesis, but only of its strand.

REAL TIME PCR : An Introduction

Although the traditional methods of quantitating mRNA are fairly good such as northern blotting and in situ hybridization, they do not
approach the ease and speed of Real Time PCR.

RT-PCR or reverse transcriptase PCR is semi-quantitative due to need to load samples on a gel and the insensitivity of
ethidium bromide. Thus, real time PCR was developed out of the need to quantitate differences in mRNA expression in a easy and
quick manner, and due to the need to use of small amounts of mRNA such as those obtained by small tissue samples, and LCM (laser
capture microdissection) isolated cells.

Real-time reverse-transcriptase (RT) PCR is different from other quantitative PCR as it quantitates the initial amount of the template
instead of detecting the amount of final amplified product (Freeman, 1999; Raeymaekers, 2000).

Real Time PCR is characterized by the point in time during cycling when amplification of the PCR product of interest is first
detected rather than the amount of the PCR product of interest which is accumulated at the end-point after PCR which contained a
large number of cycles. Real Time PCR does this by monitoring the amount of fluorescence emitted during the PCR reaction, and this
acts as an indicator of the amount of PCR amplification that occurs during each PCR cycle. Thus, in newer Real Time PCR machines,
one can visually see the progress of the reaction in "real time".

Real Time PCR also has a much wider dynamic range of up to 107-fold (compared to 1000-fold in conventional RT-PCR). The
dynamic range of an assay determines how much the target concentration can vary and yet still be quantified. This wide dynamic
range also results in a more accurate quantitation.
AFLP PCR Background

Amplified Fragment Length Polymorphism PCR, also called AFLP PCR was originally described by Zabeau et al., 1993. AFLP is
composed of 3 steps:

1-A) Cellular DNA is digested with one or more restriction enzymes. Typically this involves a combination of two restriction
enzymes: a 4 base cutter (MseI) and a 6 base cutter (EcoRI).

1-B) Ligation of linkers (restriction half-site specific adaptors) to all restriction fragments.

2-A) Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences.

2-B)

3) Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern. The aim of this tool is to
perform a theoretical AFLP-PCR experiment by using the same principles, and to suggest the adaptors and primers needed in the
experiment.

Applications of AFLP PCR

AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping
individuals for a large number of loci using a minimal number of PCR reactions.

What is Colony PCR?

The definition of Colony PCR is:

Colony PCR the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Selected colonies of bacteria
or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix
or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of
interest.

PCR Cycling Conditions:

First Denaturing Step: 95 °C , 5 min

Denaturing Step: 95 °C, 30 sec

Annealing Step: 50-55 °C, 30 sec

Extension Step: 72 °C, 1min/kb

Repeat Steps 2-4: 34 Times

Final Extension: 72 °C, 3 min

Load the entire PCR reaction onto 1% agarose gel.

Inverse PCR Background

Inverse PCR also called IPCR, and was first described by Ochman et al. in 1988 (1).
A limitation of standard PCR is that 5' and 3' flanking regions of your DNA fragment of interest must be known. Inverse PCR allows
you to conduct PCR when you only have the information of one internal sequence.

Inverse polymerase chain reaction is a variant of PCR, and is used when only one internal sequence of the target DNA is known. It is
therefore very useful in identifying flanking DNA sequences of genomic inserts. Similar to other PCR methods, inverse PCR
amplifies target DNA using DNA polymerase.

Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usual
orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle.

The Inverse PCR Method

The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This
cut results in a known sequence at either end of unknown sequences.

Inverse PCR Steps

1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion.
2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation
product.
3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence
generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction).
4) Standard PCR is conducted with primers complementary to the now known internal sequences.

In summary:

Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to
generate circular DNA.

PCR primers pointing away from the known sequences are then employed to amplify the flanking sequences.
Applications of Inverse PCR

Inverse PCR has numerous applications in molecular biology including the amplification and identification of sequences flanking
transposable elements, and the identification of genomic inserts.

Reverse Transcription Polymerase Chain Reaction

Reverse transcription polymerase chain reaction (RT-PCR) is based on the polymerase chain reaction (PCR). More importantly it is
based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses.

The techniques of RT-PCR allows the formation of cDNA (complementary or copy DNA) from RNA, which stores the sequence of
RNA (such as messenger RNA, mRNA) in the more stable form of nucleic acid, DNA. This reverse transcription from RNA into its
reverse complement DNA (cDNA) is the first step of a usually two-step process of RT-PCR. Furthermore, by copying the RNA into
DNA, one can then amplify the cDNA sequence by using primers specific for the DNA sequence. This amplification is the final
second major step of the two-step process of RT-PCR.

The Process of RT-PCR

The First step of RT-PCR is referred to as the "first strand reaction". In the first-strand reaction, complementary DNA also termed
cDNA, is made from the messenger RNA template of interest using oligo dT (oligonucleotide poly-dTs act similar to primers and bind
to the 3' polyA sequence located at the 3' UTR - untranslated region, which are present in most mRNAs), dNTPs, and an RNA-
dependent DNA polymerase, reverse transcriptase, through the process of reverse transcription.

These factors are combined in a reverse transcriptase buffer for 1 hour at 37°C. After reverse transcriptase reaction is complete, and
the cDNA has been synthesized, RNaseH is added (an RNA digestion enzyme) which digests the RNA away from the RNA-cDNA
hybrid. After incubation with RNaseH, standard PCR or polymerase chain reaction is conducted using DNA oligo primers specific for
the sequence of interest. This second step is referred to as the "second strand reaction".

Thus by adding the thermostable DNA polymerase, upstream and downstream DNA primers, the single stranded DNA becomes
double stranded and is amplified, allowing the detection of even rare or low copy mRNA sequences by amplifying its complementary
DNA.

Applications of RT-PCR

The exponential amplification of complementary sequence of mRNA or RNA sequences via reverse transcription polymerase chain
reaction allow for a high sensitivity detection technique, where low copy number or less abundant RNA molecules can be detected. It
is also used to clone mRNA sequences in the form of complementary DNA, allowing libraries of cDNA (cDNA libraries) to be
created which contain all the mRNA sequences of genes expressed in a cell. Furthermore, it allows the creation of cDNA constructs
which were cloned by RT-PCR and allow the expression of genes at the RNA and protein levels for further study.

Assembly PCR Protocol

Assembly PCR is a polymerase chain reaction variation that artificial synthesizes long DNA sequences by performing PCR
on a pool of long oligonucleotides (primers) with short overlapping segments. The oligonucleotides alternate between sense and
antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final
long DNA product.

Asymmetric PCR Protocol

Asymmetric PCR is used to preferentially amplify one strand of the target DNA more than the other.

Applications of Asymmetric PCR

The Asymmetric PCR is useful in some sequencing and hybridization probing applications where having only one of the two
complementary stands is sufficient or required.
Asymmetric PCR Method

The asymmetric PCR method is conducted as the standard pcr protocol, however a great excess of the primers for the chosen strand is
used.

Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are
required.

Also see Linear After the Exponential PCR or LATE-PCR.

Primers for Asymmetric PCR

A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations.

As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher
concentration primer continues to primer synthesis, but only of its strand.

REAL TIME PCR : An Introduction

Although the traditional methods of quantitating mRNA are fairly good such as northern blotting and in situ hybridization, they do not
approach the ease and speed of Real Time PCR.

RT-PCR or reverse transcriptase PCR is semi-quantitative due to need to load samples on a gel and the insensitivity of
ethidium bromide. Thus, real time PCR was developed out of the need to quantitate differences in mRNA expression in a easy and
quick manner, and due to the need to use of small amounts of mRNA such as those obtained by small tissue samples, and LCM (laser
capture microdissection) isolated cells.Real-time reverse-transcriptase (RT) PCR is different from other quantitative PCR as it
quantitates the initial amount of the template instead of detecting the amount of final amplified product (Freeman, 1999; Raeymaekers,
2000).

Real Time PCR is characterized by the point in time during cycling when amplification of the PCR product of interest is first
detected rather than the amount of the PCR product of interest which is accumulated at the end-point after PCR which contained a
large number of cycles. Real Time PCR does this by monitoring the amount of fluorescence emitted during the PCR reaction, and this
acts as an indicator of the amount of PCR amplification that occurs during each PCR cycle. Thus, in newer Real Time PCR machines,
one can visually see the progress of the reaction in "real time".

Real Time PCR also has a much wider dynamic range of up to 107-fold (compared to 1000-fold in conventional RT-PCR). The
dynamic range of an assay determines how much the target concentration can vary and yet still be quantified. This wide dynamic
range also results in a more accurate quantitation.