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Sequencing

Deoxyribonucleic Acid (DNA) contains genetic information in the form of base sequences of nucleotides that produce amino acids in protein formation. There are four types of bases that are complementarily paired: adenine is paired with thymine by two hydrogen bonds whilst cytosine is paired with guanine by three hydrogen bonds. DNA sequencing is a process that determines the precise base sequence of DNA using various methods. Knowledge on base sequences is integral in the biological sciences in understanding how genotypes affect phenotypes, formation of genetic disorders and fundamental genome information. The first sequence was produced by chromatography in the 1970s, however due to advancements in technology various methods have been developed to determine base sequences quickly and accurately. There are four main types of DNA sequencing: Sanger method, Maxam-Gilbert method, primer walking and shotgun sequencing.

The Sanger or Dideoxy method uses the Polymerase Chain Reaction (PCR) to produce strands of parent DNA with free nucleotides with dideoxynucleoside triphosphate (ddNTP) attached to the bases. Once a nucleotide with a ddNTP is used in the new strand it is truncated. Upon completion of the PCR, a mixture of differently truncated fragments of parent strand is produced which are then separated by gel electrophoresis. The parent strand sequence can be determined by organizing the lengths of strands.

The Maxam-Gilbert method uses chemicals to cleave at specific nucleotides where each base can be isolated. The fragments are then radioactively labeled, and undergo gel electrophoresis to be separated. Using x-rays the DNA fragments can be displayed in dark bands, which then base sequence is determined.

Primer walking uses larger portions of DNA, larger than 800 base pairs, which can be sequenced by using primers in the Sanger method. Shotgun sequencing cuts target DNA portions into small a size, which then is sequenced and arranged by identifying overlapping portions – this method relies heavily on computer analysis and control. 

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