Senin, 17 Juli 2017

Meaning and Principle Electrophoresis and Polymerase Chain Reaction (PCR)

Photo by Hal Gatewood on Unsplash - 

Electrophoresis is a method that used to separate purify macromolecules, especially proteins and nucleic acids based on charge.

Principle of electrophoresis :
  • Separation DNA, RNA, and protein caused electrical field.
How to Detect separated DNA

By using gel electrophoresis that is separation for mixing material with different charges. The gel usually use cross linked polymer.

It is usually used a polycrylamide gel, made from different concentrations of acrylamide and substances that allow cross-linking, and the result is some nets of polycramide in different cavity size.

Nucleic Acid Sequencing

Nucleic acid sequencing is methods for determining the order of the nucleotides bases adenine, guanine, cytosine and thymine in a molecule of DNA.

The Sanger DNA sequencing method
  • Uses dideoxy nucleotides to terminate DNA synthesis.
  • DNA synthesis reactions in four separate tubes
  • Radioactive dATP is also included in all the tubes so the
  • DNA products will be radioactive.
  • Yielding a series of DNA fragments whose sizes can be measured by electrophoresis.
  • The last base in each of these fragments is known.
Next Generation Sequencing
  • Illumina (Solexa) sequencing
  • Roche 454 sequencing
  • Ion torrent: Proton/PGM sequencing
  • SOLiD sequencing
Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work.

Principle of Polymerase Chain Reaction PCR

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions copies of a particular DNA sequence

The principle of PCR is a technique or method of multiplication (replication) of enzymatic DNA operates without the use of organisms

PCR process consists of three stages :
  • Denaturation: A DNA fragment (doubel strand) was heated at 950⁰C for 1-2 minutes so it will separate into single chain (single strand).
  • Annealing: Then do annealing (annealing) at a temperature of 550⁰C for 1-2 minutes, the oligonucleotide primer attached to the template DNA sequences complementary to the primer.
  • Amplification: After the settlement, the temperature was raised to 720⁰C for 1.5 minutes. At this temperature, the DNA polymerase enzyme will do the process polymerase, the DNA chain that will form a hydrogen bridge with the template DNA. This process is called amplification (amplified/copied several times)
Reactions such as above can be repeated up to 25-30 times (cycle) thus obtained DNA molecules into new double chain.

The number of amplification depends on the concentration of the target DNA to be used.

How to detect the PCR product

The product of a PCR should be a fragment or fragments of DNA in defined length.

Techniques to detect PCR product reviews they are:
  • Agarose gel and/or polyacrylamide gel electrophoresis
  • Restriction endonuclease digestion
  • Dot blots
  • High-pressure liquid chromatography
  • Electrochemiluminescence
  • Direct sequencing
The simplest and commonly used technique is electrophoresis of the PCR product on an agarose gel with EtBr (ethidium bromide).

EtBr is a fluorescent dye that intercalates into the DNA. Size markers can be electrophoresed on the gel to allow size determination of the PCR product.

Application of PCR:
  • Detection of DNA or RNA, and viruses are difficult to detect, such as HIV
  • Detection of genital human papilloma virus
  • Testing the quality of drinking water
  • Determination of nucleotide sequence
  • Knowing an alliance between species or to find out where the species come.
  • Keep track of one's origin by comparing the "finger print“
  • The diagnosis of genetic disorders before birth
  • Forecasting hemophilia
  • Isolation of DNA from mushroom mycelium and spores
*Sumber: Poppy D, Sodaya U, Lisa J, Shulhan Zalil A
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