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.
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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