melting temperature of DNA Chargaff's rule on DNA & RNA denaturation renaturation hyperchromism hypochromism

1. Melting
temperature of
DNA
Mandira S. Bhosale . MSC-Part1
FMBCH2122005
Melting temperature of
DNA
Mandira S. Bhosale.
MSc-part 1 sem 2
FMBCH2122005

2. Nucleic acid:
•DNA
•RNA

3. DNA structure:
• Double helix, Polymeric molecule , unit of
heredity & organized into genes , contains
genetic information.
• DNA – deoxyribonucleic acid
• Made up of nucleotides.
• 3 parts of nucleotide:
1) 5 carbon sugar; deoxyribose
2) phosphate group
3) nitrogen base (A, T, G,C)
• Backbone consist phosphate group and
sugar , bases sticking out of the backbone.

4. RNA structure:
• Single stranded
• RNA – ribonucleic acid
• Creates proteins via translation
• Have three main types:
1) mRNA
2) tRNA
3) rRNA
• 3 parts of nucleotide:
1) 5 carbon sugar; ribose
2) phosphate group
3) nitrogen base (A, U, G,C)
• Backbone made of alternating sugar(ribose) and
phosphate groups.

5. Types of bases:
DNA:
Purines:- adenine and
guanine
Pyrimidines:- Cytosine
and thymine
RNA :
purines:- adenine and
guanine
Pyrimidine:- Cytosine
and uracil

6. Base pairing:
• Each side of the DNA strand forms a
"complementary" hydrogen bond to the
other side.
• A must always opposite to a T.
• C must always opposite to a G.
• There is hydrogen bonding between the
bases. 2H-bonds between A and T and
3H bonds between G and C.
• In RNA A pairs with U and C pairs with G.

8. Chargaff's rule:
• The composition of DNA from many different organisms was.
analyzed by E.Chargaff and his colleagues.
• It was observed that concentration of thymine was always
equal .to the concentration of adenine (A = T). And the
concentration of cytosine was equal to the concentration of
guanine (G=C).
• This strongly suggest that thymine and adenine as well as
cytosine and guanine were present in DNA with fixed
interrelationship.
• Also the total concentration of purines (A +G) always equal
to the total concentration of pyrimidine (T +C). All DNA
possess purine and pyrimidine in equal proportions (1:1
ratio).
• However, the (T+A)/ (G+C) ratio was found to vary widely in
DNAs of different species.

9. A + G
1
T + C
■lf (A +T) > (G + C) then DNA is
referred as (AT- type).
■lf (G + C) > (A +T) then DNA is
referred as (GC- type).

10. Effect of Chargaff's rule on RNA:
• A = U
G = C
• A+G C+U
• Single stranded molecule
• Base pairs are not present in paired
forms.
• Secondary structure: double stranded in
only some regions between.
• Does not contain equal ratio.
• Does not follow Chargaff's rule.

11. Problems:
 If A = 20% in particular double stranded DNA molecule , what are the percentage % of
T ,G, C?
SOLUTION=
If A is 20% then according to Chargaff's rule T will be also 20% because they must equal
A=T
So A + T are 20% + 20% = 40%
Assuming 100% ,subtract the A & T amounts from 100. ( 100% – 40% )= 60%
60% is the amount of C & G together , so 60% / 2 = 30%
So, C is present 30%
G is present 30%

12. Problems:
 In one polynucleotide strand of DNA molecule ,the ratio of A+T/ G+C = 0.3 . What is
the A+ G/T+C ratio of the entire DNA molecule?
SOLUTION=
A + T/G + C ratio is species specific and here mentioned to be 0.3.
While A + G/T + C is the ratio which is constant for all double-stranded DNA and its value
is 1.
Because in a ds-DNA Adenine will always bind to Thymine so A=T while Guanine will
always bind to Cytosine so G=C and
Purine will always be equal to pyrimidines according to Chargaff rule.
So, the correct answer to the question is '1'.

13. Denaturation:
• Loss of helical structure of DNA called
denaturation.
• In living cell, DNA denature or separates during
replication by protein helicases .
• In laboratory, DNA strands can be separated by,
1. change pH – alkaline
2. Heat – increasing the temperature increases
the denaturation
How to measure the denaturation?
By spectrophotometer ,
denatured DNA absorbs more light than
stacked DNA. (hyperchromism)

14. Tm of DNA:
Melting temperature of
DNA
Temperature at
which half of the
DNA molecules are
denatured .

15. Factors affecting Tm of DNA:
1. Nucleotide content of DNA molecule
2. Length of DNA molecule.
3. Ionic strength of the DNA solution.

16. 1. Nucleotide content of DNA molecule
• In DNA, A pairs with T with 2 hydrogen bonds,
G pairs with C with 3 hydrogen bonds.
• G C base stacking interactions are most stable.
• Tm of DNA is greatly influenced by GC content of nucleotide.
• Example:
1.
GC Rich
2. AT Rich

17. Higher the GC
content higher the
Tm of DNA

18. 2. Length of DNA molecule.
Longer the
length of DNA
molecule higher
the Tm of DNA
More the length greater the stabilizing
forces between two DNA strands

19. 3. Ionic strength of the DNA solution.
Higher the ionic
strength of
solution tends to
higher Tm of
DNA.
>

20. Renaturation:
• Also known as annealing .
• Separated complementary strands of DNA can spontaneously re-associate to
form double helix.
• Temperature of DNA lowered below its melting temperature.
• Rewinding of DNA takes place.
• DNA can melt and reanneal itself reversibly.
• Absorbance decreases and viscosity of the solution increases.( hypochromism)

21. References:
• https://www.biotechfront.com/2021/01/what-are-chargaffs-rules.html
• https://www.toppr.com/guides/biology/molecular-genetics/chargaffs-rule/
• https://www.quora.com/Does-the-Chargaff-rule-apply-for-a-double-stranded-
RNA
• https://www.youtube.com/results?search_query=tm+of+dna
• https://www.youtube.com/watch?v=XtXfHclIrxg
• Voet and voet.
• Harper’s illustrated biochemistry 26th edition.
• Cell and molecular biology by Gerald Karp.