Characterization of Nucleic acids by Physical methods:

Isolation of DNA or RNA

Separation of Nucleic acids: •By chromatography•By electrophoresis•By Centrifugation

Characterization of Nucleic acids by Chemical methods:Sequencing of DNA

Molecular Cloning

Nucleic Acid Fractination:

DNA and RNA should be purified from tissues or cells in for their identification, characterization or manipulation.

Nucleic acids are tightly associated with proteins.

After lysis of the cells the mixture is extracted with phenol and/or iso-amyl alcohal and choloform mixture. The proteins are precipitated and rmain at the interphase, and necleic acids are in aquous phase.

After three to four time extraction as described above, the aquous phase is free of any protein. The DNA from the aquous phase can be precipitated by 70% ethanol in presence of sodium acetate salt at low temperature.

Alternatively, cellular extract can be subjected to proteinase K digestion to get rid of all proteins.

To get DNA only from the nucleic acid mixture, the samples are treated with DNase-free Rnase to get rid of RNA.

To isolate RNA, the mixture can be treated with DNase.

In order to keep the nucleic acids intact, nuclease inhibitors like chelating agents and specific inhibitors are used during isolation and extraction proceedures.

Chromatography: Hydroxyapetite Chromatography

mRNA isolation by affinity chromatography

Hydroxyapetite chromatography: (a solid phase containing calcium phosphate)

Very useful for DNA fractionation. Cell lysate is passed through HA column followed washing the column with low strength phosphate buffer to get rid of RNA and proteins.

Single and double stranded DNAs are eluted by low and high concentration of phosphate buffers (double stranded DNA binds more tightly to HA than single stranded DNA).

Thermal Chromatography: Elution of specific DNA from HA column by melting the DNA by heating and using low strength phosphate for elution.

Affinity chromatography for mRNA purification: Eukaryotic mRNAs are synthesized with a poly A stretch at their 3’ end. An oligo-dT conjugated column is used to purify mRNA.

Separation of Single and double-strended DNA by Hydroxyapetite column chromatography

Separation of Nucleic acid by Agarose gel Electrophoresis:

Polyacrylamide gels used for short length nucleic acid molecules.

0.8-2% agarose is used for medium to long range molecular weight DNA and RNA

Pulsed field Gel Electrophoresis is used for separating very large DNAs (chromosomal DNAs)

DNA bands on the gels are detected by staining the gel with intercalating dyes which flouresce after binding with double stranded DNA.

Sourthern blotting: Identification of a specific sequence of DNA on the gel using a complimentary probe.

Northern blotting: Identification of a specific sequence of RNA on the gel using complementary DNA probe.

Sourthern Blotting:

Run the DNA on agarose gel and denature the DNA bands with 0.5M NaOH

Transfer the DNA bands to Nitrocellulose membrane by blotting method orelectrotransfer

Vaccum dry the nitrocellulose membrane at 80 degree Celsius to permanently fix the DNA onto NC membrane

Put the membrane in hybidization buffer containing radioactive probe followed by washing and exposure of NC to X-ray film.

DNA seperation by Ultracentrifugation: Sepearation of single stranded to double stranded DNA (singlr strended is a bit denser), plasmid DNA from genomic DNA, Satellite DNA from Chromosomal DNA.

Supercoiled DNA:

Some of the circular DNAs have peculiar Supercoiled or supertwisted form some times referred as tertiary structure of DNA.

Linking Number (L): The number of times that one DNA strand winds around the other.

Twist: (T): Number of complete revolution that one polynucleotide strand makes about the duplex axis.

Writhing Number: Number of turns that a duplex axis makes about the superhelix axis.

Intercalating agents control supercoiling by unwinding DNA

Topoisomerases:

These enzymes controle the supercoiling of DNA in living systems to provide the correct topological state of DNA.

Type-I topoisomerasees: Act by creating a transient single strand breaks in DNA and then sealing it.

Type II topoisomerases: Also known as DNA gyrases, they act by cutting both strands of a duplex, passing the duplex through the break and resealing. Thus they catalyse the stepwise negative supercoiling of DNA at the expense of ATP hydrolysis.