Cot curve, melting temperature, unique and repetitive DNA

Cot curve, melting temperature, unique and repetitive DNA

1. MELTING TEMPERATURE, COT
CURVE AND UNIQUE AND
REPETITIVE DNA
Submittedto: Submittedby:
Dr.AryaP.Mohan KrishnapriyaM
Asst.Professor Roll.No:10
St.Teresa’sCollege,Ernakulam 1
st
M.Sc.Botany
St.Teresa’sCollege,Ernakulam

2. DNA STRUCTURE
•Double helix, polymeric molecule, unit of heredity and organized into
genes, contains genetic information.
•DNA –deoxyribonucleic acid.
•Made up of nucleotides.
•3 parts of nucleotide:
a) 5 carbon sugar; deoxyribose
b) phosphate group
c) nitrogen base (A, T, G, C).
•Backbone consist phosphate group and sugar. Each phosphate groups in
DNA strand carries –vecharge. These negative charges on each strand
repel each other.

3. HOW DO TWO STRANDS OF DNA STAY TOGETHER
Histones are positively charged and helps in compaction of DNA. Their
positive charge neutralize the negative charge of phosphate groups. 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.
•Denaturation is measured by spectrophotometer ( denatured DNA absorbs
more light than stacked DNA.

5. MELTING TEMPERATURE OF DNA(T m)
•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 Melting Temperature (Tm).
•Primers with Tm in range of 52-58℃ produce best results.
•Primers with Tm above 65℃ have tendency for secondary annealing.

7. •Tm can be calculated using Guanine-Cytosine content of a sequence.
4 ( G + C) +2 ( A + T) =℃
•Primer should optimally contain 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
bonding of G and C bases.

8. FACTORS AFFECTING Tm
Melting temperature depends on variety of factors such as
❖ Nucleotide content of DNA molecule
❖ Length of DNA
❖ Ionic strength

9. 1. NUCLEOTIDE CONTENT OF DNA MOLECULE
•InDNA,AdeninepairswithThyminewithtwohydrogenbonds.
•GuaninepairswithCytosinewiththreehydrogenbonds.
•G-Cbasestackinginteractionsaremoststable.
•TmofDNAisgreatlyinfluencedbyG-Ccontentofnucleotide.
Eg:5’ACTGCAGTGCGATCCAGCATGATC 3’
3’TGACGTCACGCTAGGTCGTACTAG 5’
ThisisanexampleofG-Crichnucleotidesequence.

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 solution tends 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% depending on 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 complementary strands of DNA can spontaneously re-associate
by cooling to form double helix is known as renaturation.
•Renaturationled to the discovery of repetativeDNA.
•Temperature of DNA lowered below it’s melting temperature.
•Rewinding of DNA takes place.
•DNA can melt and reannealitself reversibly.

14. EXPERIMENT
•Double stranded DNA are heated.
•This results in denaturation of 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 technique for measuring complexity (size of DNA or genome).
•Based on principle of DNA renaturationkinetics.
•It also gives the percentage of single stranded DNA present after
denaturation.
•During renaturation, there are repetitive as well as unique sequence in
DNA.
PRINCIPLE:Rate of renaturationis directly proportional to concentration of
complementary sequence (number of times a sequence has been
repeated in a genome).

16. •PROCESS:DenaturationofDNAbyheatingandallowedtoreannealby
cooling.LargeDNAmoleculestakelongertimetoanneal.
•CALCULATION
Cot=initialDNAconcentration(Co)×time(t)×bufferfactor
(moles/litre) (sec)(accountsforeffectof
cations).
•IndsDNA,aftercompletedenaturationandattimeofrenaturation,ifa
solutionhasthousandsofmultiplesequence,chanceofrenaturation
increases.
•Ifonlytensequencearepresent,chanceofrenaturationdecreases.

17. Thus in brief,
•More repetitive sequence in DNA increases renaturationand unique
sequence decreases 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 temperature so it reannealvery
quickly, thus time is less.

18. •Large cot ½ value = sequence are at low concentration so it reannealvery
slowly, thus more time.
•Cot ½ = initial concentration of 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): homologous DNA fragments that are
present in multiple copies in the genome.
b) Moderately repetitive sequence (MR):Short sequences that are repeated
10-1000 times in the genome.
c) Unique/single copy sequence (SL): Unique sequence of 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 hundred nucleotides at their longest).
•Present in clusters in which sequence repeats itself over and over again
without interruption.

21. •A sequence arranged in this end-end manner is called tandem.
REPETITIVE DNA
2 types:
a) Interspersed elements: usually present as single copies and distributed
widely throughout genome.
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 sequence above 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 sequence repeats : If the flanking region of a sequence is constant at
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 sequence is a variable
at both it’s terminals and only the middle region is constant.
eg: CACACA GCCGATC CACACA
(V) (C) (V)

26. •If a tandem repeat region contains many repeats a mechanism called
forward replication slippage may occur.
•During replication, when DNA is single stranded, the repeat region forms
loops.
•The DNA polymerase may accidently skip this looped region and as a
result replicated strand contains a decreased number of tandem repeats.

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

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 clusters containing as many as 3000 repeats.
•It occupy considerably shorter stretches of 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
paternity cases.
•Tandem repeats occur in DNA when a pattern of one or more nucleotides is
repeated and repetitions are directly adjacent to each other.
Eg: AATTTTCCGGCCCCAAAATTCC AATTTTCCGGCCCCAAAATTCC
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 clusters of about
10-40 bp in length which are scattered quite evenly through genome.
•DNA replicating enzymes have trouble copying regions of genome that
contain these small, repetitive sequence, which cause stretches of DNA to
change in length through generation.
•Because of their variable lengths within population, microsatellite DNAs is
used to analyze relationships between different human population.

33. TYPES OF MICROSATELLITE DNA S
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 Molecular Biology. Himalaya
Publishing House (India) Pvt.ltd.
2. Karp, G. ( ). Cell Biology (6
th
edt).

35. THANK YOU