Cot curve, melting temperature, unique and repetitive DNA

1. MELTING TEMPERATURE, COT
CURVE AND UNIQUE AND
REPETITIVE DNA
Submitted to: Submitted by:
Dr. Arya P. Mohan Krishnapriya M
Asst. Professor Roll. No: 10
St. Teresa’s College, Ernakulam 1st M.Sc. Botany
St. Teresa’s College, Ernakulam

2. DNA STRUCTURE
• Doublehelix, polymeric molecule, unit of heredity and organized into
genes, containsgenetic information.
• DNA – deoxyribonucleicacid.
• Made up of nucleotides.
• 3 parts of nucleotide:
a) 5 carbon sugar; deoxyribose
b) phosphategroup
c) nitrogen base (A, T, G, C).
• Backboneconsist phosphategroup and sugar. Each phosphategroups in
DNA strand carries –ve charge. These negative charges on each strand
repel each other.

3. HOW DO TWO STRANDS OF DNA STAY TOGETHER
Histones are positively charged and helpsin compaction of DNA. Their
positive charge neutralizethe negative charge of phosphategroups. Thus two
strands of DNA stay together.

4. DENATURATION
• Loss of helical structure of DNA into single strands through heat is called
denaturation.
• In laboratory, DNA strands can be separated by
a) change PH – alkaline
b) Heat – increasing the temperature, increases denaturation.
• Denaturationis measured by spectrophotometer ( denaturedDNA absorbs
more light than stacked DNA.

5. MELTING TEMPERATURE OF DNA(Tm)
• All primers in reaction must have similar melting temperatures.
• So that they anneal to and dissociate from complementary DNA sequence
at approximate same temperature.
• The temperature at which one half of DNA duplex will dissociate to
become single stranded is called MeltingTemperature (Tm).
• Primers with Tm in range of 52-58℃ produce best results.
• Primers with Tm above 65℃ have tendencyfor secondaryannealing.

7. • Tm can be calculatedusing Guanine-Cytosine content of a sequence.
4 ( G + C) +2 ( A + T) =℃
• Primer should optimallycontain 40-60% G-C content.
• G-C content =number of G’s and C’s in primer as a percentage of total
bases.
• Presence of G or C bases within last five bases from 3’end of primer is
called G-C Clamp.
• G-C Clamp helps to promote specific binding at 3’end due to stronger
bondingof G and C bases.

8. FACTORS AFFECTING Tm
Melting temperaturedependson variety of factors such as
❖ Nucleotidecontent of DNA molecule
❖ Length of DNA
❖ Ionic strength

9. 1. NUCLEOTIDE CONTENT OF DNA MOLECULE
• In DNA, Adenine pairs with Thymine with two hydrogen bonds.
• Guanine pairs with Cytosine with three hydrogen bonds.
• G-C base stacking interactionsare most stable.
• Tm of DNA is greatly influenced by G-C contentof nucleotide.
Eg: 5’ACTGCAGTGCGATCCAGCATGATC 3’
3’TGACGTCACGCTAGGTCGTACTAG5’
This is an example of G-C rich nucleotide sequence.

10. 2. LENGTH OF DNA
• Longer length of DNA molecules, higher will be Tm of DNA.
• More length, greater will be stabilizing forces between two DNA strands.
• More heat energy is required to dissociate DNA strands, thus Tm will be
high.

11. 3. IONIC STRENGTH
• Higher ionic strength of solutiontends to have higher Tm of DNA.

12. • DNA melting is measured by absorbance of UV light (260nm) by DNA
solution.
• Amount of UV light absorbed is proportional to fraction of non-bonded
base pair.
• As temperature increases, melting of double stranded DNA is initiated and
absorbance of UV light increases.
• Absorbance increases by 30-40%dependingon DNA sample.
• The middle point of temperature range over in which strands of DNA
separate gives the melting temperature of DNA (Tm).

13. RENATURATION
• Also known as annealing.
• Separated complementarystrands of DNA can spontaneouslyre-associate
by cooling to form doublehelix is known as renaturation.
• Renaturationled to the discovery of repetative DNA.
• Temperature of DNA lowered below it’s melting temperature.
• Rewinding of DNA takes place.
• DNA can melt and reanneal itself reversibly.

14. EXPERIMENT
• Doublestranded DNA are heated.
• This results in denaturationof DNA.
• The solution is now cooled and DNA fragments re-associate called
renaturation.

15. COT CURVE
• It is developed by Roy Britten and Eric Davidson in 1960.
• It is a techniquefor measuring complexity (size of DNA or genome).
• Based on principleof DNA renaturationkinetics.
• It also gives the percentage of single stranded DNA present after
denaturation.
• During renaturation,there are repetitive as well as uniquesequence in
DNA.
PRINCIPLE: Rate of renaturationis directly proportional to concentrationof
complementarysequence (number of times a sequencehas been
repeated in a genome).

16. • PROCESS:Denaturationof DNA by heating and allowed to reanneal by
cooling. Large DNA molecules take longer time to anneal.
• CALCULATION
Cot = initial DNA concentration(Co) × time (t) × buffer factor
(moles/litre) (sec) (accountsfor effect of
cations).
• In ds DNA, after complete denaturation and at time of renaturation, if a
solution has thousands of multiple sequence, chance of renaturation
increases.
• If only ten sequence are present, chance of renaturationdecreases.

17. Thus in brief,
• More repetitive sequence in DNA increases renaturationand unique
sequencedecreases renaturation.
• If repetitive sequence is high, renaturationoccurs in less time.
• Less repetitive sequence takes longer time to reanneal.
Thus, from above equation:
low cot value = greater number of repetitive sequence.
high cot value = less number of repetitive sequence.
• Now a days, cot ½ has replaced cot.
• Cot ½ = cot at which half of DNA has reannealed.
• Small cot ½ value = sequence are at high temperatureso it reanneal very
quickly, thus time is less.

18. • Large cot ½ value = sequence are at low concentrationso it reanneal very
slowly, thus more time.
• Cot ½ = initial concentrationof DNA (Co) × ½ time (t/2)× buffer factor
(moles/litre) (sec)

19. From the above graph, three regions are studied:
a) Highly repetitive sequence (HR): homologousDNA fragments that are
present in multiplecopies in the genome.
b) Moderatelyrepetitive sequence (MR): Short sequences that are repeated
10-1000times in the genome.
c) Unique/single copy sequence (SL): Unique sequenceof DNA and codes
for protein and are sites of transcription.

20. UNIQUE AND REPETITIVE DNA
• All human share about 99% of their DNA.
• Remaining 1% accounts for variability between us- unless you are identical
twin.
REPETITIVE DNA SEQUENCE
• Consist of sequence present in atleast 10^5 copies per genome.
• Constitute anywhere from 1-10%of total DNA.
• They are short ( a few hundrednucleotidesat their longest).
• Present in clusters in which sequence repeats itself over and over again
without interruption.

21. • A sequencearranged in this end-end manner is called tandem.
REPETITIVE DNA
2 types:
a) Interspersed elements: usuallypresent as single copies and distributed
widely throughoutgenome.
b) Tandem arrays : i) Satellite
ii) Minisatellite
iii) Microsatellite

23. SATELLITE DNAs
• Highly repetitive DNA sequence.
• Mainly found in centromere and telomere of heterochromatin.
• Consist of short sequenceabove 5-500bp in length.
• It forms very large clusters in discreate areas such as centromere.
• Each cluster contains several million base pairs of DNA.
• In many species, base composition of these DNA segments is sufficiently
different from bulk of DNA that fragments containing sequence can be
separated into a distinct ‘satellite band’ during density gradient
centrifugation.
• Satellite DNAs tend to evolve very rapidly, causing sequence of these
genomic elements vary even between two closely related species.

25. TWO TERMS TO REMEMBER
a) Simple sequencerepeats : If the flanking region of a sequenceis constantat
both it’s terminals and only the middle region is the variable.
eg: _GCCT CACACACACA GCGCG_
(C) (V) (C)
b) Intersimple sequence repeats: The flaking region of a sequenceis a variable
at both it’s terminals and only the middle region is constant.
eg: CACACA GCCGATC CACACA
(V) (C) (V)

26. • If a tandemrepeat region containsmany repeats a mechanism called
forward replicationslippage may occur.
• During replication,when DNA is single stranded, the repeat region forms
loops.
• The DNA polymerase may accidentlyskip this looped region and as a
result replicatedstrand containsa decreased number of tandemrepeats.

28. • During replication,the DNA polymerase copies the templateand
sometimes stutters in areas where tandemrepeats are located.
• As a result the number of repeats increases. This is called backward
replicationslippage.

29. MINISATELLITE DNAS
• The term was coined by Jeffrey.
• Also known as VNTR (Variable Number Tandem Repeats ).
• It is an array of tandem repeats range from about 10-100 bp in length.
• Commonly found in euchromatin regions ( telomeres and centromeres) of
chromosome.
• Found in sizable clusterscontainingas many as 3000 repeats.
• It occupyconsiderablyshorter stretchesof genome.
• Highly rich in G-C region.
• Tend to be unstable and number of copies of a particular sequence often
increases or decreases from one generation to next as result of unequal
crossing over.

30. • Length of a particular minisatellite locus is highly variable in population,
even among members of same family.
• Because they are so variable in length, they form basis for technique of
DNA finger printing, which is used to identify individuals in criminal or
paternitycases.
• Tandem repeats occur in DNA when a pattern of one or more nucleotides is
repeated and repetitions are directlyadjacent to each other.
Eg: AATTTTCCGGCCCCAAAATTCCAATTTTCCGGCCCCAAAATTCC
AATTTTCCGGCCCCAAAATTCC.
These shows variations in length between individuals and number of elements
in a given region may also vary = VNTR.

32. MICROSATELLITE DNAS
• The term was coined by Litt and Lutty.
• Also known as STR (Short Tandem Repeats)and rich in A-T region.
• It is array of very short repeats found in euchromatin regions of vertebrate,
insect and plant chromosome.
• Shortest sequence(1-5 bp long) and are present in small clustersof about
10-40 bp in length which are scattered quite evenly through genome.
• DNA replicating enzymes have troublecopying regions of genome that
contain these small, repetitive sequence, which cause stretchesof DNA to
change in length through generation.
• Because of their variable lengths within population, microsatelliteDNAs is
used to analyze relationshipsbetween different human population.

33. TYPES OF MICROSATELLITE DNAS
a) Based on repeat pattern:
i. Perfect: _CACACACACACACACACACACA_
ii. Imperfect: _CACACACACA_CACACACACACACACA_
b) Compound:_CACACACACACACA CATACATACATA CATACATACATA_
c) Complex: _CACACACACACACACA_
AATAATAAT AATAAT AATAAT_
d) Based on number of base pairs:
i) Mono : CCCCCC or AAAAAA
ii) Di : CACACACACA
iii) Tri : CCA CCA CCA CCA
iv) Tetra : GATA GATA GATA GATA

34. REFERENCES
1. Upadhyay, A., & Upadhyay, K. (2005).Basic MolecularBiology. Himalaya
Publishing House (India) Pvt. ltd.
2. Karp, G. ( ). Cell Biology (6th edt).

35. THANK YOU