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Embee Diagnostics
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PCR | |
Polymerase Chain ReactionPolymerase
Chain reaction popularly known as PCR is one of the most powerful
Diagnostic tools Modern Science has ever invented. Based on DNA detection
of a specific Bacteria or Virus, or a genetic disorder as of today PCR is
the ultimate in Human Diagnostics. It is due to the invention of PCR only
that Human genome mapping became possible. The
techniques which have been used earlier like ELISA, RIA are no doubt good
for screening but have got their own Limitations also e.g. false
positivity & negativity.
However in PCR all these are ruled out due to specific detection of
diseases specific DNA. Deadly Diseases like Tuberculosis which are
very difficult to diagnose in early stages can be detected by PCR just on
the onset of the diseases. Not only this, the biggest advantage of
detecting M.Tuberculosis by PCR is the fact that all the False positives
are ruled out which is today a major problem
with the existing detection methods like ELISA. E.g. a TB ELISA kit
has a sensitivity & specificity of just about 70% whereas the
sensitivity & specificity of PCR
is 100%. The
detection by PCR helps in two ways .i.e. - Early detection -
Confirmed
detection. Early
detection helps the patients to be put on drugs at the earliest so that
the patient has to be put on the therapy for the least possible period
thereby saving the cost of drugs as well as the Mental agony of the
patient. Confirmed
detection helps patient not being subjected to the therapy which is not
required at all. In
cases of Viral diseases like HBV, HCV or HIV, not only does PCR help in
confirmed detection but the Viral loads can be monitored for the patients
on therapy thereby helping the physician understand whether the patient is
responding to a particular drug or not which is just simply not possible
by other methodologies. PCR
detection is not limited to only the Bacterial & Viral diseases. All
the genetic disorders
or Chromosomal aberrations can be detected by PCR besides a whole lot
of other application in HLA etc. The
technique has gained popularity in the International Scenario due to
various benefits
offered by the technology. There is an ever growing demand for investigations
based on PCR & in future
majority of the tests wherever there is a possibility of getting a DNA
will be performed by PCR only. PCR
has three basic steps: n
EXTRACTION
OF DNA n
AMPLIFICATION
OF DNA n
DETECTION
OF DNA. DNA
EXTRACTION: The
extraction process in the past involved very tedious procedures & was
a limiting
factor for diagnostic PCR. But with the advancement in technology the
extraction procedures have become so simple that now the DNA can be
extracted from any kind of sample within 20-60 minutes with some simple
reagent addition and Centrifugation e.g. a kit like DNA-Prepmate-M. The
entire proceedings of the test and accuracy of the tests depend upon a
good extraction system used.
AMPLIFICATION
OF DNA: After
DNA is extracted, it can be amplified to over a billion copies from even
one DNA with the aid of Thermal Cycler. This is how it becomes possible to
detect the DNA as the number of copies now are enough to be detected.
There are several reagents etc. required for amplification like specific
Primers, Taq, dNPTS, premixes led to a total simplification of the process
as well as contamination control in the methodology. DETECTION
OF DNA: Detection
of DNA can either be done by an Electrophoresis system or a liquid Hybridization
depending upon the fact that if a simple detection and confirmation is
required then it could be done by Electrophoresis also but in case of
quantification, a liquid hybridization is necessary. INSTRUMENTS
REQUIRED FOR EXTRACTION of DNA
& RNA: For
DNA extraction a Digital Non-refrigerated centrifuge of at least 1300 rpm with RCF of about 16000g. A good and
reliable centrifuge is a must because DNA extraction involves steps which
are sensitive to the accurate speed & RCF For
RNA extraction, a Digital Refrigerated centrifuge of 14000 rpm with RCF of
about 15000-16000 g. A good and reliable refrigerated centrifuge is a must
because RNA is sensitive to temperature & the extraction involves
steps which are sensitive to the speed & RCF. A
good autoclavable set of Micro-pipettes is required for DNA or
RNA extraction in the range of 2-20ml, 20-200ml,
200-1000ml,. A
variable speed of say 0-3000rpm Vortex Mixer is required
which should have a heavy duty construction. A
microwave oven for Lysis of the cells will also be required. A
water bath for boiling & incubations. INSTRUMENTS
REQUIRED FOR AMPLIFICATION OF DNA OR RNA: Thermal
Cycler is the instrument which can amplify one DNA to almost a
billion copies. In the initial stages the amplification of DNA was done by
robotic use with the help of several water baths. The invention of Thermal
Cycler led to a total revolution in PCR. Today the amplification of DNA is
just possible within 2 hours with the help of a thermal Cycler.
Amplification process involves three steps i.e. -- Denaturation
of DNA at 96C.
-- Anneling of
Primers at 56 C.
-- Extension of
primers at 72 C. All these steps are performed by a Thermal Cycler. A
good autoclavable set Micro-pipettes is required for DNA or
INSTRUMENTS
REQUIRED FOR DETECTION OF AMPLIFIED PRODUCT: After
the amplification process for detection based on the methodology used, for
detection
following Instruments are required: Gel Electrophoresis System: A small Gel electrophoresis system which is capable of providing a consistent 50 or 100 volt current. Should be flexible enough to accommodate small or large gels. UV TRANSILLUMINATOR: This instrument is required for Visualization of the bands which will be generated by electrophoresis. GEL
DOCUMENTATION SYSTEM: This system along with the ELISA READER: This is required for those PCR assays which are Based on Liquid hybridization for the calorimetric estimations. There
are few other instruments during the process for sample processing as well
as for the preparatory work e.g.
SPACE
REQUIRED FOR PERFORMING PCR. Three
clean rooms of approximately the size of 6x 6 feet are enough. Two rooms
preferably should have a small Anti-room type of an opening but is not
essential. PARAMETERS
AVAILABLE IN PCR: These days availability of parameters by PCR is not a problem & a whole lot of parameters as available. Click here to go back to our featured section We would appreciate
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