History Of Polymerase Chain Reaction

Posted on December 15, 2011 by admin
History Of PCR

History Of PCR

The polymerase chain reaction is a new test type discovered these last decades. Polymerase chain reaction is discovered by Kary Mullis, a scientist working for a new company in biotechnology field. Its primary job was to make short DNA chains so other scientists in the company lab could work over them.

One day while he was thinking about any method to analyze mutant methods of DNA he discovered a new way to be able copying any region of any DNA. This way he could be able to amplify the DNA. He didn’t recognize in the begging what he had discovered but lately he knew that he made a big step forward in bio-molecular science field and got Nobel Prize in Chemistry in 1993. He wrote an article in the famous Journal Scientific American and there he noted that by using polymerase chain reaction method he would be able to manufacture 100 billion similar molecules of genetic DNA material in a matter of time. Also we wrote in this article that polymerase chain reaction method it’s not only faster, but it’s much easier because to do this kind of test the only things you need are heat source, centrifuge, few simple reagents that can be found in any lab and  test tubes.

Information that was included in this article is that for a polymerase chain reaction test the DNA segment does not need to be pure, it can be isolated simply before testing and can be used in PCR without problem. This made the polymerase chain reaction test famous and used in every lab that has to do with genetics and biogenetics. Nowadays polymerase chain reaction test is used to locate viruses and bacteria, to find DNA foot printings, and even cloning different type of cells.

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How To Isolate DNA For The PCR Test Works

Posted on December 15, 2011 by admin
pcr-test

PCR Test

PCR test comes in few test types. By this test you can analyze nucleic acids that are included in RNA or DNA. But DNA is more used in PCR tests because it’s more stable than RNA and molecules in DAN can be isolated easier than these molecules in RNA. Only a single DNA simple is enough for a polymerase chain reaction test.

The most important step in polymerase chain reaction is to make it sure that your DNA you are going to test should not be contaminated with other DNA’s and it have to be isolated very well. For isolating the DNA you can follow the steps bellow.

  1. You can Obtain Cells that contain DNA from fingernails, swabbing in the inside walls of the mouth or from hair roots. See the forth step…
  2. Centrifuge these cells you obtained at 1200- 1500Xg for approximately 5 minutes. After this you can resuspend these cells in 1 ml PBS in the same centrifuge speed, 1200- 1500Xg for approximately 5 minutes. After repeating wash these cells in distilled water. In this step the only thing you must be aware is cell debris’s. if this occur see step four.
  3. Now you can place the DNA container in boiling water again for approximately 5 min. this way you can deactivate the DNAse molecules that can be found in centrifuge and other preparation steps. After this concentrate the sample with ethanol alcohol and in this way the sample will be ready for PCR test.

Usually this kind of PCR test is done twice or 3 times to be sure and provides the control of the quality. And this test is important because mostly its used for HIV control.

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The Three Steps of Polymerase Chain Reaction

Posted on December 15, 2011 by admin
PCR Steps

Step Of PCR

Polymerase chain reaction has been considered as one of the most important breakthroughs of the 20th century in the field of molecular biology and genetics, despite only being around only since the 1980s. In this process, it is now possible for minute quantities of genetic material to be amplified, which in turn, would enable researchers to identify and come up with entirely different DNA; look for infectious organisms, including harmful viruses and elements that cause hepatitis, AIDS, and TB, among others; and even detect variations in genetic makeup, including mutation of genes in human beings.

There are three steps involved in a polymerase chain reaction: denaturation, annealing and extension.

Denaturation occurs in genetic material, and it is the step wherein DNA molecules, which are double stranded, are converted into single strands of DNA.   In the second step, the primers are annealed to the regions of the molecules that are now single-stranded.   On the last step, which is extension, the molecules are extended, thanks to the DNA polymerase’s action.

These three steps are subject to specific temperatures: with denaturation, temperature must be at 94 degrees Celsius; with annealing, it must be at 60 degrees Celsius; and for extension, it must be at 70 degrees Celsius.  If these specific temperatures are not followed, the steps will not be successful and the PCR process will not take place.

Aside from PCR, another process called RT-PCR also occurs. RT-PCR stands for reverse transcription polymerase chain reaction, a process wherein PCR occurs, and is followed by the conversion of the sample DNA into a complementary DNA with  enzyme reverse transcriptase, which means DNA goes back into being a double-stranded DNA.

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Reverse Transcription PCR

Posted on December 15, 2011 by admin
Reverse Transcription PCR

RT-PCR

Reverse transcription PCR is a technique used mostly to find out and quantifying mRNA. There are also more fields where Reverse Transcription PCR is used like cDNA library construction , in cloning cells, reverse transcriptions (like its name) and also for amplifying synthesized cDNA. There are many reagents used in reverse transcription PCR technique and each of them has their purpose.

For example:

  • mRNA is used to create templates for DNA generation process.
  • Reverse transcriptase reagent is used to convert mRNA into cDNA, so the cDNA library can be created.
  • DNA primers are specific to the mRNA and this help to span the right sequence of DNA.
  • Buffer RT-PCR is like a maintainer for reverse transcriptase and the Taq DNA polymerase.
  • Deoxynucleoside triphosphates are used by reverse transcriptase and Taq DNA polymerase and use to generate the cDNA.
  • Taq DNA polymerase is a heat resistant and in this way it can be used as DNA polymerase that synthesizes DNA.

By having knowledge what is capable to do any reagent we can proceed to reverse transcription PCR step by step.

  • In the first step, the RTC reverse the mRNA template to a similar ssDNA. While this is happening the DNA primers and the TRC is connected to the mRNA template so the DNA can be replicated.
  • In the temperature of 55°C  DNA primers checks out the new synthesized ssDNA’s.
  • In the temperature of 72°C Taq DNA polymerase enter the reaction and this way the ssDNA is extended. 72°C is the optimal temperature for Taq DNA polymerase to operate.

Now that cDNA molecule is created the reverse transcription PCR continues like below:

  • To 94°C temperature the cDNA is denaturized.
  • To 55°C temperature DNA primers anneal to the new cDNA.
  • To 72°C temperature the Taq polymerase is included in reaction and DNA is extended.
  • These steps are repeated over and over depends on the amount of DNA you are looking to create.
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Some Facts About Polymerase Chain Reaction

Posted on December 15, 2011 by admin
polymerase chain reaction

PCR

The process of polymerase chain reaction or PCR is a fairly new discovery, its history spanning for only close to three decades.  In 1983, Kary Mullis was working for Cetus, a pioneer company in the field of biotechnology, located in Emeryville, California, when he invented PCR.  His work in the company was to manufacture short DNA chains which will be used by other scientists.  Mullis, in a written account, still remembers thinking of PCR while riding his motorcycle along the Pacific Coast Highway 128.  He was thinking of new methods of analyzing mutations that happen in DNA when his thoughts veered into a method of copying or amplifying any region of the DNA.  His thoughts paid off, as he did not only invent a breakthrough in genetics, he was also awarded the Nobel Prize in chemistry ten years later, in 1993.

Mullins wrote in the journal Scientific American that starting with just a single molecule of the genetic DNA material; the polymerase chain reaction he invented has the capacity to generate as much as 100 billion similar molecules in a matter of hours.  Moreover, he asserted the convenience of the process he invented by saying that the amplification of DNA in PCR is not only faster than the other process of cloning, but also that it is easier to execute because the only requirements are test tubes, a heat source and a number of “simple reagents.”

Moreover, in order to do PCR, the original segment of DNA that a person wants to copy does not need to be pure or present in abundant amounts. It can be pure, but if it is a part of a mixture of certain materials, the DNA can still be used for PCR. The convenience of PCR has found the process playing a crucial role in various uses.  PCR has become an indispensible tool for any laboratory that studies genetic material.  Polymerase chain reaction is now used in the diagnosis of genetic diseases, in DNA fingerprinting, in locating viruses and bacteria, in the study of human evolution, in establishing biological (e.g. paternal) relationships, and even in cloning an Egyptian mummy DNA, among others.

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