Sequencing of Nucleic Acids

Sequencing of DNA and RNA was challanging before 1975. Only few RNA samples were sequenced using snake venom phosphodiestrase or pancreatic phosphodiesterase. These enzymes remove nucleotide residues from 3 and 5 ends of RNA respectively.

The free nucleotides are seperated and identified by cgromatography.

But the discovery of Restriction endonucleases, development of DNA sequencing techniques and molecular cloning have made it possible to sequence and DNA in hours to days time period.

Restriction Endonucleases (REs):

These are DNA cutting enzymes, which recognize specific base sequence of 4-8 bases in double stranded DNA and cleave both strands of duplex.

Type I and III Restriction endonucleases: They have both

endonuclease and methylase activity on a single protein. Type I REs cleave the DNA at random site located at 1000 base pairs from the recognition site. Type III RE do the same at 24-24 bp away from recognition sites.

TypeII RE: They cleave DNA at specific site within the recognition
sequence. This property has made type II RE as most important tool in molecular biology. There are more than 200 different sequence specificity belonging to more than 2000 enzymes.

Many of RE recognize Palindromic DNA sequences.

When they cut the two strands which are symmetrically staggered about the center of the palindromic recognition sequence, they yield restriction fragments with complimentary single stranded ends with one to four nucleotides in length. These types of ends are referred as cohesive or sticky ends. Some RE cut the recognition sequences through the twofold symmetry producing restriction fragments with fully base paired blunt ends.

Restriction maps provide a mean of characterizing a DNA molecule.

Restriction-Fragment Length Polymorphism (RFLP) is used as a marker for a gene and it is very useful to detects mutation in genes.

DNA fingerprinting: Slight sequence differences that occur from individual to individual every 500 to 1000 bp is referred as Sequence polymorphism. These differences alter the restriction site for RE and therefore, when digested with same RE, a different restriction length fragments are obtained. These patterns are unique to each individual and referred as DNA finger print. This is the most powerful technique to identify an individual with just a small amount of blood, semen, hair, skin or any tissue sample. Total DNA is digested with the same RE and separated on an agarose gel. The DNA is then transferred to nylon membrane and subjected to southern blotting using a common probe.

Techniques for Sequencing of DNA:

Chemical method: Developed by Maxam and Gilbert 1. Chain terminator procedure also known as dideoxy method developed by Fredrick Sanger. The Chemical Procedure: Once the DNA is cleaved by specific RE to smaller fragments. Then each fragment is sequenced as follows

I.

II.

II.

Labeling of the 5 end of DNA with radio active phosphate Removal of 5 phosphate by alkaline phosphatase Labeling of 5 end by polynucleotide kinase in presence of g-32P labeled radioactive ATP. Cleavage of DNA in a base-specific manner and different fragments created are resolved on hi resolution polyacrylamide gel (gel should be able to resolve fragments with 1 bp difference). By exposing the gel to X-ray films an autoradiogram is obtained. The position of a particular base in the DNA is identified by the relative position on the gel of the corresponding radioactive

fragment.

Chemical sequencing: G specific reagents: Dimethyl sulphate followed by treatment with piperidine. A+G Specific cleavages: Same as above except the first step in acidic condition. C specific cleavage: Treatment of DNA with hydrazine in 1.5M NaCl followed by piparidine treatment.

C+T specific cleavages: Treatment of DNA with hydrazine followed by piperidine treatment.
Please refer to Fig. 28.55 and 28.56 in the text book (Voet)

Sequencing by Chain termination method; the dideoxy procedure:

In this method, DNA polymerase I is used to make a complimentary copy of the single stranded DNA being sequenced. DNA polymerase I requires a template (the DNA to be sequenced acts as template), a short primer strand with free 3end and the dNTPs. Four reactions are carried out with radio-labeled or fluorescent-labeled primer and mixture of three dNTPs and dideoxy analogue (ddNTP)of either dATP ot dCTP or dGTP ot dTTP in each reaction mixture. As the new chain elongates, it gets terminated when ddNTP is incorporated in the chain. All the fragments are seperated on a gel and autoradiographed. Sequence is read from all the four reaction fragments and their length.