LECTURE NOTES ON GENE SEQUENCING

GENE SEQUENCING

 DNA sequencing is a method of  sequencing for determining the order of the nucleotide bases - adenine, guanine, cytosine, and thymine - in a molecule of DNA of an organism. Knowledge  of DNA sequences has become important for basic biological research and in numerous applied fields like  diagnostic service,biotechnology,forensic biology and Taxonomy.

The  basic unit of the genome is a single deoxyribo nucleotide.  It is a complex process.DNA sequencing involves the determination of the order of DNA bases.

Sanger sequencing("Next- Generation“ sequencing)

It is method to find out the nucleotides Sequence of unknown DNA strand. Sanger sequencing has been known as as "Next- Generation“ sequencing methods, especially for large scale genome analyses and for obtaining especially long DNA sequence reads (>500 nucleotides).

This method generally is an In-Vitro synthesis of DNA strand and by using terminators (di-deoxynucleotide) the growing strand terminates at specific site.Upon termination the strands are overlap to got original sequence of unknown DNA Strand.

REQUIREMENTS FOR SANGER SEQUENCING

Single Stranded DNA  template

Primer

DNA polymerase enzyme

Di-Deoxynucleotides:The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs).Every nucleotide have its specific ddNTP form i.e., ddATP, ddGTP etc

Procedure

1. Denaturation of DNA

2. Primer attachment and extension of bases

3. Termination of Sequence

4. Gel electrophoresis for separation of DNA fragments

The double stranded DNA template is treated with heat so that it becomes single stranded.A short, single-stranded radioactively labeled primer  is added to the end of the DNA templateThe  template DNA and primer are added in four seperate tubes.Then  ddNTPs were added in tubes in the way that single tube contain one type of ddNTP.Extension is start and band of various sizes are formed.The DNA fragments  are separated by electrophoresis.Overlap these sequences to find out sequence of Target DNA.

 

Maxam–Gilbert sequencing

Maxam–Gilbert DNA sequencing is a method of sequencing developed by Allan Maxam and Walter Gilbert in 1976–197.Maxam–Gilbert sequencing was the first widely adopted method for DNA sequencing, and, along with the Sanger dideoxy method.method based on chemical modification of DNA and subsequent cleavage at specific nitrogenous bases.

PRINCIPLE

It involves the purification of the DNA fragment that to be sequenced and labeled with radioactive material.Chemical treatment generates breaks at a specific nitrogenous bases and thus a series of radio labelled fragments is generated. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation.These fragments are  visualize in X-ray for autoradiography. Procedure

This sequencing utilises  radioactive labeling at one 5′ end of the DNA fragment to be sequenced (gamma-32P).Chemical treatment generates breaks at a small proportion of one or two of the four bases of nucleotide in each of four reactions (G, A+G, C, C+T). For example,

1. the purines (A+G) generated by using formic acid,

2. the guanines and to some extent the adenines are generated  by dimethyl sulfate,

3. the pyrimidines (C+T) generated  by using hydrazine.

4. NaCl add to hydrazine for generating Cytosine.

Each chemicals are then added in separate tube to generate series of labeled fragments .Electrophorosis of the fragments in the four reactions are done  side by side for size separation.To see the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each showing the location of identical radiolabeled DNA molecules.

APPLICATIONS:

By DNA sequencing  one  can understand the function of a specific sequence and the sequence responsible for any disease.

By  comparative DNA sequence study we can detect any mutation.

It is used in DNA fingerprinting.By DNA sequencing the human  genome sequence, Human genome project get completed.

In forensic study DNA sequencing is used to identify particular individual because every individual has unique sequence of his/her DNA. It is particularly useded to identify the criminals by finding some proof from the crime scene in the form of hair, nail, skin or blood samples.

In agriculture DNA sequencing used  in mapping and sequencing of the whole genome of microorganisms has allowed the agriculturists to make them useful for the crops and food plants.

In medical research, DNA sequencing can be used to detect the defective  genes which are associated with some heredity or acquired diseases. Researchers  use different techniques like gene therapy to identify the defected genes and replace them with the healthy ones.