Inverse polymerase chain reaction (Inverse PCR) is a variant of the polymerase

chain reaction that is used to amplify DNA with only one known sequence. One
limitation of conventional PCR is that it requiresprimers complementary to both
termini of the target DNA, but this method allows PCR to be carried out even if only
one sequence is available from which primers may be designed.

Inverse PCR is especially useful for the determination of insert locations. For
example, various retrovirusesand transposons randomly integrate into genomic
DNA. To identify the sites where they have entered, the known, "internal" viral or
transposon sequences can be used to design primers that will amplify a small portion
of the flanking, "external" genomic DNA. The amplified product can then be
sequenced and comparedwith DNA databases to locate the sequence which has
been disrupted.

The inverse PCR method involves a series of restriction digests and ligation,
resulting in a looped fragment that can be primed for PCR from a single section of
known sequence. Then, like other polymerase chain reaction processes, the DNA is
amplified by the temperature-sensitive DNA polymerase:

1. A target region with an internal section of known sequence and

unknown flanking regions is identified
2. Genomic DNA is digested into fragments of a few kilobases by a
usually low-moderate frequency (6-8 base) cutting restriction enzyme.
3. Under low DNA concentrations, self-ligation is induced to give a
circular DNA product.
4. PCR is carried out as usual, with primers complementary to sections
of the known internal sequence.*

Finally the sequence is compared with the sequence available in the data base.

 Note: although the figure suggests that the circularized ligation product is
digested prior to PCR, this is not the case. PCR does not require linear products
and the use of another restriction enzyme to cut the known sequence could also
cut within the unknown region, resulting in a failed PCR.
Allele-specific PCR is a varation of the polymerase chain reaction which is
used as a diagnostic or cloning technique, to identify or utilize single-
nucleotide polymorphisms (SNPs) (single base differences in DNA). Allele-
specific PCR does require the sequence of the target DNA sequence, including
differences between the alleles.

Primers for Allele-Specific PCR

The allele-specific PCR uses primers whose 3' ends encompass the SNP.
PCR amplification under stringent conditions is much less efficient in the
presence of a mismatch between template and primer, so successful
amplification with a SNP-specific primer signals presence of the specific SNP
in a sequence.

Reverse transcription polymerase chain reaction (RT-PCR) is a variant

of polymerase chain reaction (PCR), a laboratory technique commonly used
inmolecular biology to generate many copies of a DNA sequence, a process termed
"amplification". In RT-PCR, however, an 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 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 exponential
amplification [1].

RT-PCR includes three major steps. The first step is reverse transcription (RT), in
which RNA is reverse transcribed to cDNA using reverse transcriptase. This step is
very important in order to perform 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 DNA extension from the primers. This is done
with thermostable Taq DNA polymerase, usually at 72°C, the temperature at which
the enzyme works optimally. 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, whereas the
amplicon can be visualized only after the amplification ends.

Real-time RT-PCR provides a method in which the amplicons can be visualized as

the amplification progresses using a fluorescent reporter molecule. There are three
major kinds of fluorescent reporters used in real time RT-PCR, which are 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 an 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]Useof reverse transcription polymerase chain

reaction
The exponential amplification via reverse transcription polymerase chain reaction
provides for a highly sensitive technique in which a very low copy number of RNA
molecules can be detected. RT-PCR 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 analysis is used to study the RNA's gene expression
further. RT-PCR can also be very useful in the insertion of eukaryotic genes into
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 or 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.

Drug Classification:
Drugs can be classified according to various criteria including chemical
structure or pharmacological action. The preferred classification is the latter
one which may be divided into main groups as follows:

a) Chemotherapeutic agents - used to cure infectious diseases and cancer.

(Sulfa drugs, Antibiotics)
b) Pharmacodynamic agents - used in non-infectious diseases (Cholinergic,
Adrenergic, Hallucinogenic, Sedatives)
c) Miscellaneous agents (Narcotic Analgesics, Local Anesthetics)

Drug Names:

Drugs have three or more names including a: chemical name, brand or trade
name, and generic or common name. The chemical name is assigned according
to rules of nomenclature of chemical compounds. The brand name is always
capitalized and is selected by the manufacturer. The generic name refers to a
common established name irrespective of its manufacturer.

In most cases, a drug bearing a generic name is equivalent to the same drug
with a brand name. However, this equivalency is not always true. Although
drugs are chemically equivalent, different manufacturing processes may cause
differences in pharmacological action. Several differences may be crystal size
or form, isomers, crystal hydration, purity-(type and number of impurities),
vehicles, binders, coatings, dissolution rate, and storage stability.

Introduction to Drug Action

Definition:

A very broad definition of a drug would include "all chemicals other than food that affect
living processes." If the affect helps the body, the drug is a medicine. However, if a drug
causes a harmful effect on the body, the drug is a poison. The same chemical can be a
medicine and a poison depending on conditions of use and the person using it.

Another definition would be "medicinal agents used for diagnosis, prevention, treatment
of symptoms, and cure of diseases." Contraceptives would be outside of this definition
unless pregnancy were considered a disease.

Disease Classification:

A disease is a condition of impaired health resulting from a disturbance in the structure or

function of the body. Diseases may be classified into the following major categories:

1) Infections caused by viruses, ricketsia, bacteria, fungi, protozoa and worms

2) Allergic diseases caused by antigens and foreign substances
3) Metabolic disorders caused by defects in the body's ability to carry out normal
reactions - these may be hereditary, deficiency, and congenital defects
4) Cancer
5) Toxic diseases caused by poisons
6) Psychosomatic and mental diseases

Chemotherapy, broadly defined, means the treatment of any disease by chemicals

including infectious and non-infectious diseases. The original definition applied only to
drugs which were used in the treatment of infectious diseases. The proper term for the
treatment of non-infectious diseases is pharmacodynamics.
Sites of Drug Action:

l. Enzyme Inhibition:

Drugs act within the cell by modifying normal biochemical reactions. Enzyme
inhibition may be reversible or non reversible; competitive or non-competitive.
Antimetabolites may be used which mimic natural metabolites. Gene functions
may be suppressed.

2. Drug-Receptor Interaction:

Drugs act on the cell membrane by physical and/or chemical interactions. This
is usually through specific drug receptor sites known to be located on the
membrane. A receptor is the specific chemical constituents of the cell with
which a drug interacts to produce its pharmacological effects. Some receptor
sites have been identified with specific parts of proteins and nucleic acids. In
most cases, the chemical nature of the receptor site remains obscure.

3. Non-specific Interactions:

Drugs act exclusively by physical means outside of cells. These sites include
external surfaces of skin and gastrointestinal tract. Drugs also act outside of cell
membranes by chemical interactions. Neutralization ofstomach acid by antacids
is a good example.
Mode of Drug Action:

It is important to distinguish between actions of drugs and their effects. Actions of drugs
are the biochemical physiological mechanisms by which the chemical produces a response
in living organisms. The effect is the observable consequence of a drug action. For
example, the action of penicillin is to interfere with cell wall synthesis in bacteria and the
effect is the death of the bacteria.

One major problem of pharmacology is that no drug produces a single effect. The primary
effect is the desired therapeutic effect. Secondary effects are all other effects beside the
desired effect which may be either beneficial or harmful. Drugs are chosen to exploit
differences between normal metabolic processes and any abnormalities which may
be present. Since the differences may not be very great, drugs may be nonspecific in
action and alter normal functions as well as the undesirable ones. This leads to undesirable
side effects.

The biological effects observed after a drug has been administered are the result of an
interaction between that chemical and some part of the organism. Mechanisms of drug
action can be viewed from different perspectives, namely, the site of action and the
general nature of the drug-cell interaction.

l. Killing Foreign Organisms:

Chemotherapeutic agents act by killing or weakening foreign organisms such as bacteria,

worms, viruses. The main principle of action is selective toxicity, i.e. the drug must be
more toxic to the parasite than to the host.

2. Stimulation and Depression:

Drugs act by stimulating or depressing normal physiological functions. Stimulation

increases the rate of activity while depression reduces the rate of activity.