Cot curve analysis for gene and genome complexity

This Presentation will be helpful to undergraduate and postgraduate students of biology and biotechnology in understanding the significance of COT curves in determination of gene and genome complexity amoug various organisms

1. COT (C
0T) CURVE
ANALYSIS AND
GENOME/GENE
COMPLEXITY
{Renaturation/Denaturation
Kinetics}
DR.GURPREET SINGH, PH.D
ASSISTANT PROFESSOR-BIOTECH
LYALLPUR KHALSA COLLEGE, JALANDHAR (PUNJAB -INDIA)
Email: [email protected]

2. Introduction
•themorecomplextheorganism,themoreDNAis
neededto“runiti.e.totalamountofDNAinthegenome
•Therefore,wewouldexpectalinearrelationship
betweengenomesizeandorganismcomplexity.
•Atthelowerrangeofcomplexity,thisholds:
•Inlargerorganisms,relationshipbreaksdown
•Bacteriahavesmallergenomesthaneukaryotes,and
viruseshavesmallergenomesthanbacteria.Organisms
haveDNAapparentlyinexcessofwhatisneeded;
repetitivesequences,“junkDNA”.
•ThisistheCvalueParadox,thatinthemostcomplex
organisms,theredoesn’tappeartobetheexpected
relationshipbetweencomplexityandgenomesize.

3. C-value and C-Value paradox
•TheamountofcellularDNAindifferentorganisms
doesnotcorrelatewiththeirrelativebiological
complexity.
•term C-value was first introduced in 1950 by Swift &
C-value paradox by Thomas.
•SwiftH. 1950. The constancy of desoxyribosenucleic acid in plant
nuclei. Proc Natl Acad Sci USA 36:643–654.
•Thomas CA Jr. 1971. The genetic organization of chromosomes.
AnnuRev Genet 5:237–256.

4. Four variables affecting estimations of
the C-value—
the ambiguity of the term,
polyploidy,
repetitive sequences and
experimental errors—
of which polyploidy may be the most significant
Taft et al., 2007 The relationship between non-protein-coding DNA and eukaryotic
complexity. BioEssays29 :288–299,

5. Some genome sizes compared
Mycoplasma 1 x 10
6
Bacteria 1.5 x 10
6
Fungi 5x10
7
Algae 8 x 10
7
Molds 8 x 10
7
1 x 10
8
Worms 1 x 10
8
Molluscs 9 x 10
8
5 x 10
9
Insects 1 x 10
8
2 x 10
9
Echinoderms 1 –2 x 10
9
Cartiaginous
fishes 5 –8 x 10
9
BoenyFishes 5 x 10
8
1 x 10
10
Amphibians 5 x 10
8
9 x 10
10
Reptiles 2 -5 x 10
9
Mammals 3 –5 x 10
9
Birds 9 x 10
8
1 x 10
9
Flowering plants 5 x 10
7
1 x 10
11

8. What is the explanation?
•Early evidences of genome structures
came from
•DNA Denaturation-renaturation
studies

18. cot curve -Cot curve is concerned with the measurement of the
degree of reannealing of DNA strands

19. When dsDNA is denatured by heat and
allowed to reanneal it the reaction can be
described by
•dC/dt = -kC
2
•
C is the concentration of ssDNA at time = t
•
K is a rate constant
•
If the equation is integrated over C
0
at t = 0 and C after time t has passes it
becomes:
•
C/C
0
= 1
•
1 x k C
0
t
•
One useful point of reference is the point where half the DNA remains in ssDNA
form
•
C/C
0
= ½ = 1/1 x k C
0
t
½
•
Can be reduced to C
0
t
½
= 1/k , the so called Cot ½ value = product of
•
concentration and time describes the reaction to reach the half way point
•
Larger Cot
½ values reflect longer renaturation times.

20. Double stranded DNA denatures at high temperature
UV light absorbance increases as DNA goes from double-stranded to single-stranded
Melting temperature (T
m)
“Hyperchromic shift”
What is the basis of hyperchromic shift? Stacked bases absorb less UV light

21. Significance of the
C
0TCurve Analysis
1.Genome complexity
2.Gene complexity

22. Single-stranded
DNA
(denatured)
Gives high A
260
absorbance
Double-stranded DNA
(reassociated)
Gives low A
260absorbance
Cot Curve Analysis of DNA Samples
Therefore, the genome with the more unique DNA sequences has the higher Cot value.
Note: Organisms with larger genome sizes (in nucleotide pairs) usuallyhave higher Cot values
(as illustrated in the Genome Size portion of the above figure).
“Complexity” is a measure of the amount of “uniqueness” of the DNA sequences in a genome.
How to measure Cot?
By looking at the reassociationkinetics of DNA
Cot values are a measure of the “complexity” of an organism’s genome

24. What Repeat Classes Represent
•Unique DNA:
–up to 10 copies:
–about 60% of the genome
–highly conserved coding regions
–other highly conserved regions
–other non-conserved unique sequences
•Moderately repeated DNA
–average of 500 copies,
–a total of 30% of the genome
–transposon-based repeat
–large gene families
•Highly repeated DNA:
–average of 50,000 copies per genome
–about 10% of total DNA
–constitutive heterochromatin
–microsatellites
–a few highly repeated transposon families (Alu sequences)

25. Melting temperature T
m
increases with G:C content
Why? Because G-C base pairs that have 3 hydrogen bonds that require more energy to
disrupt than A-T base pairs, which have only 2 hydrogen bonds.

26. Visualizing DNA by Ethidium Bromide Staining
•DNA binding dyes such as ethidium bromide and acridine orange intercalate between the stacked bases of DNA.
•Agarose gels, such as the one shown at the top right, are used to separate different length DNA fragments.
Once the gel is stained with ethidium bromide, the stained DNA is visualized by shining UV light on the gel
(ethidium bromide gives off a bright red fluorescence under UV light!).

27. G value paradox
relationship between organismal complexity
and the number of protein-coding genes
Why this paradox?
Partofthisparadoxcanbeexplainedbyanincreased
utilizationofalternativesplicing,whichallowsa
greaterrangeofproteinisoformstobeexpressed,
whichclearlyoccursinthecomplexorganisms,althoughthisinturn
necessitatesanincreaseinregulation.

30. Theratioofthetotalbasesofnon-protein-codingtothetotalbasesofgenomicDNAper
sequencedgenomeacrossphyla(i.e.thepercentncDNA).Thefourlargestprokaryote
genomesandtwowell-knownbacterialspeciesaredepictedinblack.Single-celledorganisms
areshowningray,organismsknowntobebothsingleandmulticellulardependingonlifecycle
arelightblue,basalmulticellularorganismsareblue,plantsaregreen,nematodesare
purple,arthropodsareorange,chordatesareyellow,andvertebratesarered.