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Transcript of PCR
In 1986, Cetus scientists isolated the
, a bacterium found in
Because Taq could withstand high temperatures, it removed the need for human intervention during the reaction, streamlining and shortening the process. Without a heat-resistant enzyme like Taq polymerase, PCR could not be used on a large scale as the process would have been too slow.
Types of PCR:
WHAT is PCR?
(Polymerase Chain Reaction)
The polymerase chain reaction (PCR) is
a biochemical technology in molecular biology used to amplify a single or a few copies of a piece of DNA
across several orders of magnitude, generating
thousands to millions
of copies of a particular DNA sequence.
* the diagnosis of
*the identification of genetic fingerprint
(used in forensic science and paternity testing)
* the detection and diagnosis of
, PhD, a scientist at the Cetus Corporation, conceived of PCR as a method to copy DNA in large amounts of a specific target DNA. Over the next two years, a team of scientists that recognized the potential impact PCR could have on molecular biology, researched, refined and made the theoretical process a reality.
* PCR analyses detect specific target genes.
* PCR results are available within days.
* PCR analyses are sensitive.
* PCR analyses can be performed on a variety of sample types.
* PCR analyses can be used to survey the general microbial community or target specific genes.
• PCR results can be affected by the presence of some metals or humic acids (not common).
• Standardization of protocols for sample collection, storage, and extraction between laboratories is currently under way but is not yet complete.
• The development of PCR analysis is based on known biodegradation pathways and gene sequences.
advances have enabled PCR to become the technology it is today ,
the thermal cycler.
The device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The cycler then
the temperature of the block in discrete, pre-programmed steps.
It is a laboratory apparatus most commonly used to amplify segments of DNA via the (PCR)
Thermal cyclers may also be used in laboratories to facilitate other temperature-sensitive reactions.
(PCR machine or DNA amplifier)
1) Standard PCR.
2) Nested PCR.
3) Multiplex PCR.
4) Restriction fragment length polymorphisms (RFLP).
5) Long PCR.
6) Reverse-transcriptase-polymerase chain reaction (RT-PCR).
7) Real Time PCR.
Contamination control and PCR process repeatability is simplified with PCR Cabinets. Each PCR Cabinet purge the work area of contaminants between amplifications and during preparatory procedures.
An integrated UV lamp enables rapid decontamination of the work zone between experiments and prevents cross-contamination.
THANK YOU !
Dr. Nariman Sindi
clear steps in each PCR cycle,and each cycle approximately doubles the amount of target DNA, These are:
1. Separating the Target DNA -
the tube containing the sample DNA is heated to more than 90 degrees which separates the double-stranded DNA into two separate strands.
2. Binding Primers to the DNA Sequence -
Two primers are used - one for each of the newly separated single DNA strands. The primers bind to the beginning of the sequence that will be copied. The tube is cooled and primer binding occurs between 40 and 60 degrees celsius. This step yields two separate strands of DNA, with sequences marked off by primers. The two strands are ready to be copied.
3. Making a Copy -
The temperature is increased to approximately 72 degrees Celsius Beginning at the regions marked by the primers, nucleotides in the solution are added to the annealed primers by the DNA polymerase to create a new strand of DNA complementary to each of the single template strands.