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Identity to Organisms: DNA Sequencing
#1
DNA, our genetic material provides identity to our characters. Being the universal genetic material for higher organisms, it is almost similar in one organism. This method of identification is known as DNA sequencing which is widely used in taxonomic studies etc. DNA sequencing is different from DNA fingerprinting as it involves determining base sequence rather than comparing DNA fragments.

DNA is made up of thousands of nucleotides which in triplet codons encode specific proteins. Order of nucleotides in DNA is called the DNA sequence which is commoner in one organism. There are specific regions common to one type of organism. As the total sequence is huge, only the specific region required to identify one type of organism is sequenced for example, ITS region for fungi, 16s r RNA region for bacteria etc. These regions are amplified by using specific primers prior to sequencing. Species specific primers which are part of these regions corresponding to unique products of different species can be used to generate more accurate results.

Prior to DNA sequencing, DNA of interest is isolated, purified and amplified by Polymerase Chain Reaction (PCR). PCR results in millions of copies of the DNA fragment to be sequenced. Two methods are used in DNA sequencing; Chain termination method/Sanger Coulson Method and Chemical Degradation Method/ Maxam-Gillbert method.

Sanger Coulson method requires the DNA to be sequenced to be cloned into a vector (M13). In Chain termination method, strand synthesis is done with the help of modified DNA polymerase which is called as kleno fragment with polymerase activity. This strand synthesis involves PCR reactions and different from usual DNA synthesis as this makes use of di-deoxynucleotides which lacks 3’ Hydroxyl group. When this special type of nucleotides is used, after this nucleotide other nucleotides cannot be added which results in chain termination. Fragments of different length can be obtained. Four vials corresponding to four types of nucleotides consisting of Adenine, Guanine, Cytosine and Uracil. Only one type of dideoxy nucleotide is added to one vial. Out of the four deoxynucleotides one should be radiolabelled with P32 or S35 isotopes. In each vial, stand synthesis and amplification takes place by PCR. After the reaction, the DNA fragments are separated by gel electrophoresis. This gel is incorporated with urea which makes double stranded DNA into single stranded DNA. Since the difference in length between two fragments can be small as a nucleotide, the electrophoresis process should be well controlled. The amplified products in four vials are run separately and based on the position of the band the sequence can be determined.

In chemical degradation method, primers are not required as it doesn’t involve DNA synthesis. And this method does not require the DNA to be cloned into a vector. It involves chemicals to degrade DNA. The double stranded DNA to be sequenced is labeled with a radioactive phosphorous group at 5’ end using the enzyme polynucleotide kinase. Dimethyl sulphate is added to the labeled DNA and heated to obtain single stranded DNA. Generally assuming that one template strand contains more purines and is heavier, the other strand will move faster in a gel during separation. The amplified DNA samples are taken in four vials.
In the first vial, Dimethyl sulphate brings about a chemical modification of a specific nucleotide. It acts on Guanine and makes a nucleophillic attack 7th Nitrogen and it becomes unstable. This instability leads to breakage of DNA strand at that point. By adding piperidine, unstable Guanine is removed. Dimethyl sulphate in acidic medium is used for the second tube. This will attack purines; Adenine and Guanine, and chemically modify the bases. In the third tube, hydrazine is added along with piperidine. Hydrazine interacts with Cytosine and brings about a chemical change. Hydrazine in an alkaline medium is used for the fourth tube followed by piperidine which reacts with pyrimidines. Only a single break is made in one strand. These tubes treated with different chemicals are subjected to gel electrophoresis.

DNA fingerprinting, in contrast compare the variable number of tandem repeats (VNTR) in human DNA for identification. These are non-coding sequences of which number of repetitions is unique to a person. This is widely used in forensic investigations and paternity testing.
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#2
Pyrosequencing or 454 sequencing (named after the Roche owned company that developed the method) is a novel, single nucleotide addition method (SNA method) of DNA sequencing and is increasingly utilized by the researchers nowadays. In this method, hybridization is achieved between a sequencing primer and a PCR amplicon (single stranded) that acts as a template. Following hybridization, the DNA is incubated with the enzymes such as DNA polymerase, apyrase, ATP sulfurylase and luciferase as well as with the substrates, luciferin and APS (adenosine 5' phosphosulfate).

The deoxribonucleotide triphosphates (dNTPs) are added sequentially. The enzyme DNA polymerase, catalyses the addition of deoxyribo-nucleotide triphosphate to the strand of DNA if it forms a complementary base to the base of the template strand. During each of the nucleotide incorporation, an equimolar quantity of pyrophosphate (ppi) to the incorporated nucleotide is released in the reaction.

The enzyme, ATP sulfurylase, catalyses the conversion of PPi to ATP in the presence of the substrate adenosine 5' phosphosulfate or APS. This ATP formed drives the conversion of luciferin to oxyluciferin mediated by luciferase enzyme that produces visible light in the proportional amount to that of ATP. This small amount of visible light produced by the action of enzymes as each nucleotide is incorporated to the growing chain is detected by CCD (charge coupled device) chip and is recorded as a series of peaks referred to as a pyrogram. The peaks correspond to the order of the nucleotides that are added revealing the underlying DNA sequence and the height of each peak is relative to the number of nucleotides incorporated.

Apyrase (nucleotide-degrading enzyme) degrades the unincorporated nucleotides and ATP continuously. And when the degradation is complete, a new nucleotide is added. Thus, by means of correlating the sample flashes with the nucleotide that is present at that time period, scientists are able to sequence a stretch of DNA.


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#3
The pyrosequencing or 454 sequencing, in its commercial version can read up to twenty million bases per run by implementing the technique on picotiter plates that promotes sequencing of huge amounts of DNA at lower cost in comparison to earlier techniques. On the other hand, one most important drawback to the pyrosequencing method is incomplete extension of repeats of the similar nucleotides (such as AAAAAAA). Each read is only about 100 bp (base pairs) long and makes it hard for the researchers to distinguish among the repeated regions which are longer than this length. Still the pyrosequencing technique is improving rapidly, and latest machines are able to generate 400 bp sequence reads.

The important applications of pyrosequencing technique includes, but not limited to, identifying species and detect antibiotic resistance, quantifying DNA methylation performed as never before, gathering valuable data in a variety of studies of clinical genetic syndromes and also to achieve reliable genetic analysis in the study of a wide variety of hematological conditions. The strengths of the pyrosequencing techniques compared to other techniques is that it has built in quality control, easy communication of information, assays for any genetic marker , mutation tolerant assays, flexible range of methods, quantitative genotyping data and finally simple to use reliable platform.

DNA sequencing technologies both older and new ones have furnished humans with the information regarding many of the genomes. Starting in the 1970s, the Sanger sequencing method made it possible for the scientists to sequence stretches of DNA at faster speeds. Additional sophistication and automation of this process continued to enhance sequencing rates thus allowing scientists to accomplish the most important milestones in the Human Genome Project. At present, new methods such as pyrosequencing have significantly cut down the sequencing cost and may possibly in time allow each person to have one’s information regarding the genome.
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