2. Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry Molecular Genetics and Clinical BiochemistryKSU FOURTH WEEK

3. Biochemical Properties of Genetic Material (DNA & RNA) Tropp, B; Molecular Biology genes to proteins. Jones and Bartlett Publisher. 2008

4.  1- 1 Absorption Spectra  Absorb light in ultraviolet range, most strongly in the 254-260 nm range  Due to the purine and pyrimidine bases  Useful for localization, characterization and quantification of samples

5.  1-2 Sedimentation and density Can be characterized by sedimentation velocity (Svedberg coefficient, S)  Sedimentation velocity centrifugation  Related to MW and shape Or by buoyant density  CsCl (DNA) or CsSO 4 for RNA  Sedimentation equilibrium centrifugation

6.  G-C base pairs are more dense than A-T pairs

7.  Agarose or polyacrylamide gels  DNA is negatively charged and migrates toward positive pole when placed in an electric field  Smaller fragments move through the gel matrix more quickly and therefore migrate faster per unit of time  Extremely common method for characterizing and purifying DNA fragments Including DNA sequencing procedures

8. Gel Electrophoresis Animation

9. Reassociation Fragile Supercoiling Chemical Modification Mutation Nucleic Acid Properties Denaturation Degradation

10.  Denaturation involves the breaking of hydrogen bonds Disrupts the base stacking in the helix and lead to increased absorbance at 260 nm  Hyperchomic shift  By increasing temperature slowly and measuring absorbance at 260 nm as melting profile can be generated Temperature for midpoint of denaturation is called the T m

11.  Increased G+C gives increased T m 3 vs. 2 hydrogen bonds  Increased ionic strength also increases T m

12.  Denatured DNA duplexes can reassociate with complementary strands to reform duplex Chemical reaction, rate depends upon conditions  including substrate concentration

13. C o (starting concentration) t 1/2 (time taken for half the DNA to reassociate).

14.  DNA concentration is routinely measured in micrograms per ml (mass/volume) But here the relevant concentration is copies of complementary DNA (not mass) per unit volume And this depends upon both the mass per volume and the size of the genome being studied

17.  Previous curves were for genomes generally lacking repetitive sequence regions Al or nearly all sequences present at one copy per genome  What happens to the C 0 t analyses when genomes have repetitive sequences? Single copy, middle and highly repetitive

18. 2-2 C 0 t Analyses

20. When the nucleotide sequence of one strand is known, The sequence of the complementary strand can be predicted

21.  After nucleic acids are denatured they can be allowed to reform base pairs with complementary molecules Molecular hybridization Close but not perfect match required  stringency Can involve DNA:DNA or DNA:RNA FISH, Southern transfer (blotting) and DNA microarray analyses involve hybridization

23.  FISH  Use DNA or RNA probes for hybridization Originally radioactive Now biotin and fluorescent dyes  Cells/chromosomes fixed to slide before hybridization  Can detect single copy genes

24. Objective: Identify Specific Parts of a Chromosome

25. Procedure http://FISH

26.  This image shows chromosomes with fluorescent R-bands.  The bright green dots are probes complementary to olfactory receptor homologues.  In most chromosomes these areas are subtelomeric, i.e. near the end of the chromosomes, but in chromosome 2 (bottom, left) we see that the probe has hybridized to the middle of the chromosome. A comparison with ape chromosomes shows that the human chromosome 2 is the result of an end to end fusion of two ancestral chromosomes. As a result the two subtelomeric ends became the middle of chromosome 2, which is why we get hybridization of the probe there

27.  The southern blot is used to verify the presence or absence of a specific nucleotide sequence in the DNA from different sources  And to identify the size of the restriction fragment that contains the sequence

28. (2) Southern Blot (Animation)(Animation), (Procedure) (Procedure) (Animation)(Procedure)

29. Pipetting Pouring Mixing  The great lengths of DNA molecules make them extremely susceptible to breakage by the hydrodynamic forces Great care is taken when larger DNA molecule are isolated

30. Nature of Supercoiling  It is a physical rearrangement of the DNA double helix that allows it to conform more closely.  DNA can adopt a more compact configuration due to supercoiling.  Linking number (Lk) is the number of times the two phosphodiester backbones wrap around one another in a given distance.  It depend on the solution conditions

31.  Enzymes  DNA gyrase adds negative supercoils  DNA topoisomerases remove negative super coils  DNA replication tends to introduce positive Supercoiling

32.  Cytosine:  Adenine:  8-oxo-7,8-dihydroguanine (8-oxoG) N - methylcytosine 4 N - methyladenine 4 Deamin -ation Depuri -nation

33. Chemical Modification Alkylation (Methylation) Oxidation Hydrolysis Mutation Block Replication Remodeling DNA Repair

34.  They are hydrolytically cleave the phosphodiester backbone of DNA Nucleases 1- Endo- 2- Exo- Cleave Middle of ChainThe End of the Chain 3- Restriction Endonucleases

35.  Mutations in DNA sequences generally occur through one of two processes:  DNA damage from environmental agents such as ultraviolet light (sunshine), nuclear radiation or certain chemicals  Mistakes that occur when a cell copies its DNA in preparation for cell division.

36. Mutation Deletion Addition Translocation