A PCR machine can rise temperature upto
100 °C but after that it is not able to lower the temperature below 70 °C automatically. which step
of PCR will be hampered first in this faulty machine?explain why?
Answers
Answer:
The initial step denatures the target DNA by heating it to 94°C or higher for 15 ... At lower temperatures, PCR primers can anneal to each other via regions of ...


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PCR for the Food Microbiology Laboratory
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Rapid and automated – results usually available within 12 to 26 hours
High degree of sensitivity – able to detect one colony forming unit in a 25g sample
Precise and accurate results from very specific genetics-based detection
Flexible – assays for more than one pathogen can be run simultaneously
The most remarkable thing about the typical food microbiology laboratory is how little it has changed in the last 20 years. Time saving devices may have been introduced in that time, but the underlying culture-based technology employed would be familiar to laboratory staff in the 1980s.
This is all the more surprising when one is aware that the recent development of molecular biology has provided a whole new range of tools that have re-vitalised many branches of microbiology. Now food microbiologists too are benefiting from this revolution as some of the key molecular biology tools begin to appear in their laboratories.
Foremost among these tools are PCR (polymerase chain reaction)–based techniques that target the genetics of microorganisms, rather than their phenotypic characteristics.
What is PCR? PCR is a method for synthesising multiple copies of (amplifying) a specific piece of DNA. For PCR to take place four basic components are required:
A DNA template containing the target sequence that is to be amplified. For pathogen detection this sequence must be highly specific to the organism concerned, often a single gene, such as a virulence gene.
Primers – a pair of short single-stranded DNA sections, which are exactly complementary for specific parts of the target sequence.
A heat-stable DNA-polymerase enzyme, usually Taq polymerase from a thermophilic bacterium, which catalyses the reaction.
Free nucleotides that are used as the building blocks for multiple copies of the DNA template.
The first stage in the PCR process is to raise the temperature to about 90-95oC. This causes the double stranded DNA to denature, or melt, into single strands.
The temperature is then reduced to about 50-65oC to allow the two primers to bind, or anneal, at specific points on the single-stranded DNA of the target sequence.
Finally, the temperature is raised to 70-74oC and the DNA-polymerase enzyme catalyses the duplication of the target sequence, starting at the annealed primers on each single strand, in a process known as extension. This results in two double-stranded DNA fragments that are identical copies of the original target sequence.
The temperature cycling process is then repeated a number of times, typically 30-40, creating a theoretical doubling of the number of copies of the target sequence at each cycle. This gives an exponential increase in target DNA concentration and produces sufficient DNA for reliable detection from a single target sequence in a few hours.