Tm of DNA - melting temperature of DNA

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 – d eoxyribo n ucleic a cid 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 – r ibo n ucleic a cid 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.

7. 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 .

8. 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).

9. 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.

10. 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%

11. 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. B ecause 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'.

12. 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 )

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

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

15. 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

16. Higher the GC content higher the Tm of DNA

17. 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

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

19. 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 )

20. 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 26 th edition. Cell and molecular biology by Gerald Karp.