Physical propertiesUltraviolet absorption

Denaturation

Renaturation

Chemical properties

Nucleophiles

HydrazineHydozlyamine

Alkylating agents

Nitrous acid

Bifunctional alkylating agents

Electophiles

DNA absorbs uv in range of (240nm-280nm).

MAX absorption at 260nm.

This absorption is monitored by spectrophotometer

Calculations are done by using BEER LAMBERT LAW

A=ECl

E FOR DNA IS 6600L/MOL cm.

Absorbance(A)=1 at 260nm is equivalent to 50ug/ml DNA.

A260/A280 is a measure of DNA purity.

Single stranded DNA has absorbance 40% more then double stranded.

Absorbance at 260nm

free bases 1.60 units

single strand 1.37 units

double strand 1.00 units

When two strands of the DNA

separate as single strands.

Effect of various factors on DNA

TEMPERATURE

ACIDS

ALKALI

TM linear relationship between

Base stacking energy G+C CONTENT

Temp. at which 50% of the DNA melts

Melting temperature(Tm)

TEMPERATURE

Increase in temp. disrupts H-bond & shell

of hydration.

ACIDS

PH<2 ionizes

the bases of

two strands

Thus disrupts

H bonding &

base stacking.

Phosphodiester

bond & glycosidic

bond are susceptible

to lowPh.

ALKALI

Above ph 12

base tend to

change polarity

Disrupts H

bonding

RenaturationDenatured DNA may reform when Hbond forms btw complementary base pairs.

Slow cooling or addition of ions may lead to renaturation.

The rate limiting step in this reaction is collision of complementary DNA molecules.

Once the nucleation has initiated, rest double helix coils very fastly.

NUCLEOPHILE

Electron rich reagent that can attack on +ve lycharged carbon next to nitrogen in nitrogen bases.

H2N-NH2

It is widely used to modify DNA.

Stronnucleophile and powerfull

mutagen.

It can disrupt h bonding physically by

formation of hydroxlamino group thus

prevent A-T & C-G pairing.

Seek an unbranched electron pair.

Good site for e attack are ring nitrogen and

oxygen on nitrogen bases.

ALKYLATING AGENTS

Ethylmethylsulphonate

Ethylnitrosourea

N –methyl-N’-nitro-N-nitosoguanidino

Depending upon site of alkylation they

may alter keto enol equillibrium.

Bulky alkyl adduct on the base will affect

H bonding.

Bifunctional alkylating agents

Have two reactive group

Nitogen and sulphur mustard gas

They form intra or inter crosslinking

btw two strands of DNA

HNO2 REACTS WITH AMINO GROUP OF C,G

OR A in an oxidative deamination rx

The deamination leads to carbonyl group

cytosine

uracil

Reverse h bond polarity

adeninehypoxanthine

Reverse H bond polarity

guanine

xanthine

No mismatch pairing

Richard r sinden

Voet and voet

Cell and molecular biology

genarld karp