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DNA can undergo reversible strand separation.

DNA strand separation (denaturation) when ? how?

Natural:
Making RNA from DNA Unwinding by proteins Unwinding by DNA topology

Experimental:
Heating Low ionic strength Disruption of hydrogen bonds urea, formamide, alkaline pH

Denaturation- renaturation

Deanturation
DNA circular ? Circular DNA & denaturation a problem, why ?

Renaturation Only by complementary, Time dependent No competition if non-related seq.

Analysis of DNA denaturation


Change in light absorption (UV) 260 nm

Factors that affect Tm = Base composition pH Salt High temperature and neutral pH = ????

o. 4C

in Tm with every 1% GC

Heat denatured DNA dsDNA

quick chill ssDNA

ssDNA

ssDNA Alkaline pH neutral pH slow cooling renatured

Heat denatured

Renaturation: Two step reaction


Rate of renaturation <<<< rate of denaturation DNA hybridization: ioning strength and temperautre Salt concentraion (0.15- 0. 5M NaCl):
Too low: electrostatic repulsion between the phospate backbone Too high: neutralized the negative charge, intrastrand bonding Optimum Temperature: high enough to disrupt the random, intrastrand H bonds but not high enough to completely denature the DNA (Tm 20C)

Use: Heteroduplex mapping, Identification of different genes Relationship between different species, DNA-RNA hybridization

Renaturation kinetics and complexity


Rate of reassociation reaction depends on variety of sequences
Genome size, Gene number Larger genome more genes. Same gene appear many times ??? Different ??

What is the complexity of a genome ???


Kinetic studies

Rate of reassociation
-kC2 = dC/dt C = Concentration of single stranded DNA at time t in seconds

K = reassociation constant
Initial concentration of DNA = C0 C = C0 1 1+ k C0 t

The rate of reassociation is controlled by C0 and t


C = C0 1 2 = 1 1+ k C0 t

50% of the reaction completed , Cot Cot 1/2 = 1/k (k= reassociation rate constant)

DNA Renaturation Kinetics


The rate at which heat-denatured DNA sequences in solution will renature is dependent on DNA concentration, reassociation temperature, cation concentration, and viscosity (usually not a factor if the DNA is free of contaminants). Cot = DNA conc. (mol/L) x renaturation time in sec. x a buffer factor that accounts for the effect of cations on the speed of renaturation

Greater Cot indicates slower reaction.

The same Cot value can be reached in a variety of ways For example:

Nucleotide concentration
Renaturation time Buffer factor, 0.5 M SPB Nucleotide concentration Renaturation time Buffer factor, 0.12 M SPB

=
= X = Cot = =

0.050 M
344.000 sec 5.820 100.000 0.002 M 1.000

= X 50,000.000 sec

Cot

100.000

The total amount of reassociation for a given Cot value does not change.

The rate at which a particular sequence will reassociate is proportional to the number of times it is found in the genome.

Given enough time, nearly all of the DNA in a heat-denatured DNA sample will reassociate.

Cot curve
Fraction of the DNA that is reassociated Vs log Cot 1-C/C0 Vs log cot

Rate of reassociation and DNA length

Rate of reassociation is inversely proportional to the length of reassociating DNA

C0t amount of the DNA in the genome

Reassociation depends on the presence of complementary sequences


Eukaryotic DNA: bacterial DNA reassociation Frequency of any sequence to be +nt in eukaryotic DNA is several hundred folds less than bacterial. Therefore, concentration of eukaryotic DNA has to be in several hundred fold excess.

Rate of reassociation and DNA length


Cot is directly related to the amount of DNA in the genome
A fixed amount of DNA, 12 pg (C0 ) = 3000 E. coli (o.oo4 pg size) but only 4 eukarytoic genomes ( 3 pg size) X 750 fold in DNA concentration for specific sequence

Cot indicates the total length (bp) of different sequences = complexity

Rate of reassociation and Comlexity of the genome

A typical mammalian C0t curve has three plateaus.


When the same experiment is carried out using DNA purified from a complex eukaryote, such as human, then we do not see a simple sigmoidal curve. Instead we see a curve which is the sum of the reannealings of many different components.

C0t of the fast component = 0.25 x.0013 Complexity of the fast component = ?? Complexity of the genome =

Cot analysis
Cot analysis was first developed and utilized in the mid 1960s by Roy Britten, Eric Davidson, and associates.

It is based upon the principles of DNA renaturation kinetics.

The steps in a Cot analysis

(A) Tissue from the organism of interest is placed in an antioxidant medium and homogenized in a blender. The homogenate is filtered, plastids are preferentially lysed, and nuclei are pelleted by centrifugation. (B) The pellet should be free of contaminating organelles (as determined by phasecontrast microscopy). DNA is isolated from purified nuclei using phenol/chloroform extractions coupled with proteinase and RNase digestions.

Sonication

Check the average Dissolve in different salt solutions fragment size, ppt Dispense fixed amount in glass ampules, seal

(C) The DNA is cut into pieces of about 450 bp by sonication or high-speed blending. (D) Fragment size is checked by agarose gel electrophoresis. Sheared DNA is precipitated, and aliquots of the DNA are dissolved in 0.03 M, 0.12 M, and 0.50 M sodium phosphate buffer (SPB) to produce solutions of known concentrations. (E) The DNA solutions are distributed into glass microcapillary tubes or glass ampoules so that each tube/ampoule contains 100 g of DNA. The ends of the tubes/ampoules are sealed.

0.03 M SPB +90C Tm-25C

(F) One of the tubes containing a known concentration of DNA is selected. (G) The tube is placed in boiling water to denature DNA duplexes. (H) The tube then placed in a water bath set at the samples criterion (i.e., Tm - 25C). Renaturation is allowed to occur until the sample reaches a specific Cot value [Cot value = the product of the sample's nucleotide concentration (moles of nucleotides per liter), its reassociation time in seconds, and an appropriate buffer factor based upon cation concentration]. (I) Once the sample has reached the desired Cot value, the end is broken off of the tube/ampoule, and the solution is blown into a 100-fold excess of 0.03 M SPB.

(J) The diluted sample quickly is loaded onto a hydroxyapatite (HAP) column equilibrated with 0.03 M SPB. At this buffer concentration, all DNA binds to the HAP. (K) Once all of the solution has entered the HAP, 0.12 M SPB is added causing single-stranded DNA (ssDNA) to elute. Eluant containing ssDNA is collected in a graduated polypropylene tube. (L) After the ssDNA had been collected, 0.50 M SPB is added to the column to elute doublestranded DNA (dsDNA).

(M) The volumes of the ssDNA eluant and the dsDNA eluant are determined. (N) Exactly 0.9 ml of the centrifuged ssDNA eluant is mixed with 0.1 ml of aqueous 10 N KOH to denature any DNA duplexes. A sample of the dsDNA eluant is likewise denatured. (O) The A260 values (adjusted for light scatter at 320 nm) of the ssDNA/KOH mixture and the dsDNA/KOH mixture are determined. For a particular Cot value, the percentage of ssDNA (% ssDNA) is calculated as follows: [(Vss x Ass) x 100] [(Vss x Ass) + (Vds x Ads)] = % ssDNA where Vss = total volume of single-strand fraction, Vds = total volume of double-strand fraction, Ass = A260 (adjusted for light scatter) for the KOH-denatured single-strand fraction, and Ads = A260 (adjusted for light scatter) for the KOH denatured double-strand fraction.

P) The logarithms of Cot values ranging from essentially no renaturation to nearly complete renaturation are plotted against corresponding % ssDNA values to yield Cot points.

A graph of Cot points ranging from little or no reassociation until reassociation approaches completion is called a Cot curve. Because a DNA sequence reassociates at
a rate that is directly proportional to the number of times it occurs in the genome, sequences that occur more than once in a genome (repetitive DNA) reassociate at lower Cot values than sequences found only once per genome (single-copy DNA). From an analysis of a Cot curve one can determine genome size, relative proportions of single-copy and repetitive sequences, the fraction of the genome occupied by each frequency component, and the mean kinetic complexity of the sequences in each frequency component.

The reassociation kinetics of this eukaryotic DNA show three types of components (indicated by the shaded areas). The arrows identify the Cot1/2 values for each component. In the calculation of the complexity, E. coli DNA was assumed to have a Cot1/2 = 4. Thus the compexity of the slow component (unique DNA with a repetition frequency of 1) = (0.45) (630) (4.2 x 106 bp)/4 = 2.98 x 108 bp. The total size of the genome = 2.98 x 108 bp/0.45 = 6.6 x 108 bp.