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Amplification-based dna analysis methods

Various methods can be used for obtaining sequences of DNA, which are useful for studying disease-causing organisms. With the advent of rapid sequencing technology, our knowledge base of the entire genomes of pathogenic organisms has grown phenomenally. We start with a description of the polymerase chain reaction, which is not a sequencing method but has allowed researchers and clinicians to obtain the large quantities of DNA needed for sequencing and other studies. The polymerase chain reaction eliminates the dependence we once had on cells to make multiple copies of DNA, achieving the same result through relatively simple reactions outside the cell.

Polymerase chain reaction (pcr)

Most methods of DNA analysis, such as restriction enzyme digestion and agarose gel electrophoresis, or DNA sequencing require large amounts of a specific DNA fragment. In the past, large amounts of DNA were produced by growing the host cells of a genomic library. However, libraries take time and effort to prepare and DNA samples of interest often come in minute quantities. The polymerase chain reaction (PCR) permits rapid amplification in the number of copies of specific DNA sequences for further analysis ( [link] ). One of the most powerful techniques in molecular biology, PCR was developed in 1983 by Kary Mullis while at Cetus Corporation. PCR has specific applications in research, forensic, and clinical laboratories, including:

  • determining the sequence of nucleotides in a specific region of DNA
  • amplifying a target region of DNA for cloning into a plasmid vector
  • identifying the source of a DNA sample left at a crime scene
  • analyzing samples to determine paternity
  • comparing samples of ancient DNA with modern organisms
  • determining the presence of difficult to culture, or unculturable, microorganisms in humans or environmental samples

PCR is an in vitro laboratory technique that takes advantage of the natural process of DNA replication. The heat-stable DNA polymerase enzymes used in PCR are derived from hyperthermophilic prokaryotes. Taq DNA polymerase , commonly used in PCR, is derived from the Thermus aquaticus bacterium isolated from a hot spring in Yellowstone National Park. DNA replication requires the use of primers for the initiation of replication to have free 3ʹ-hydroxyl groups available for the addition of nucleotides by DNA polymerase. However, while primers composed of RNA are normally used in cells, DNA primers are used for PCR. DNA primers are preferable due to their stability, and DNA primers with known sequences targeting a specific DNA region can be chemically synthesized commercially. These DNA primers are functionally similar to the DNA probes used for the various hybridization techniques described earlier, binding to specific targets due to complementarity between the target DNA sequence and the primer.

PCR occurs over multiple cycles, each containing three steps: denaturation , annealing , and extension. Machines called thermal cycler s are used for PCR; these machines can be programmed to automatically cycle through the temperatures required at each step ( [link] ). First, double-stranded template DNA containing the target sequence is denatured at approximately 95 °C. The high temperature required to physically (rather than enzymatically) separate the DNA strands is the reason the heat-stable DNA polymerase is required. Next, the temperature is lowered to approximately 50 °C. This allows the DNA primers complementary to the ends of the target sequence to anneal (stick) to the template strands, with one primer annealing to each strand. Finally, the temperature is raised to 72 °C, the optimal temperature for the activity of the heat-stable DNA polymerase, allowing for the addition of nucleotides to the primer using the single-stranded target as a template. Each cycle doubles the number of double-stranded target DNA copies. Typically, PCR protocols include 25–40 cycles, allowing for the amplification of a single target sequence by tens of millions to over a trillion.

Questions & Answers

what is elements
Erneet Reply
an Element is a substance that can not be further decomposed by ordinary chemical means.
vannessa
can i get notes of biochemistry
Sneha Reply
yep why not
Abdullah
what is microbiology?
Gayanjali Reply
what is malaria
Gayanjali
bios- life/living things micro- small things that cannot be seen with the naked eye In other words the study of living things that cannot be seen with the naked eye but with the help of a microscope
Coltuneac
logos- science
Coltuneac
it is 10 to the power minus 6 biology which studies life forms on those scales including metabolism , physiology, nutrition requirements
barun
what are prokarytes
NDIMUKIKA Reply
A prokaryotic is a unicellular organanism that lacks a membrane bound nucleus ,mitochondria or other any membrane bound organelle. .......
Maryam
in prokaryotic nucleus is not developed and cell organelles are absent
Harshad
how is one who is feeling blockage in the chest with mucus be diagnosed
Essy Reply
be talking sepitol every morning
Ikenna
is that all?
Ven.phumie
What is prions
Sangeeta Reply
is tuberculosis a viral infection?
hildar Reply
No. It is a Bacterial Infection.
Orooj
what are zn staining
Evelyn Reply
full meaning zeil neelson staining
kezia
By using zn stain we diagnose / examine acid fast bacilli .
Deepak
explain hw bacteria grows
Kanyago Reply
Hw does bacteria grow?
Kanyago
Binary fission
Sangeeta
three successive phase comes in bacteria growth i.e lag phase/adaptation phase ,log phase/exponential phase ,death phase when it is in fermentation process .
Deepak
introduction to virology
Dongyile Reply
is the study of virus
FADAYOMI
Virology
Sangeeta
study of virus which can be seen by using electron microscope i.e below 1micron in size . It requires a host cell to multiply own number to proliferate itself .
Deepak
what are some five benefits of studying microbiology
Joyce Reply
please I'm waiting for answers
Joyce
microbio is very important! 1.) microbes thought to have evolved into our cells 2.) they keep us healthy 3.) they can cause disease and illness 4.) good for bioremediation 5.) show evolution in real time(observable) 6.) make the earth a liveable place (make more o2 than plants)
just
What fields are interesting to study when you are doing your postgraduate degree
Noxolo
what is mean by killed vaccine?
Nasira Reply
Heat or formaldehyde inactivated live strain
Number
what is an antimicrobial
Denis Reply
what are eukaryotes
Oenen Reply
a eukaryote is an organism whose cells contain complex structures enclosed within membranes .
Dipali
has a nucleus. prokaryotes don't have a true nucleus.
Tanisha
they are micro-organisms with complex structure or have a nuclear membrane
Irene
It has true nucleus
Sangeeta
how many protons,electrons and neutrons are there in a matter
Y'NOTS Reply
this would depend what matter you're looking at, on top of other variables such as isotopes and isomers. can we have a better picture of what you're trying to identify
Nicolas

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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