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.