1. Basic Procedures for DNA analysis I) DNA isolation & purification: –Sample: nucleated cells –Principle: A- PURIFICATION STEPS: 1.Cell lysis 2.Removal of contaminants 3.Ethanol ppt.  recovery of purified DNA B- EVALUATION OF THE PURIFICATION: 1.DNA purity by A260/A280 2.Measure DNA concentration (A260) 3.Measure DNA yield Reem M. Sallam, MD.PhD.

2. II) Restriction Endonuclease Enzymes: Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

3. Procedure & Products: Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

4. III- Denaturation (melting) of ds DNA: 1.Loss of Hydrogen bonds 2.Unstacking of bases –Each strand remain intact (intact PDE bonds) –Achieved by: 1.Increase temp. or 2.Decrease salt conc. Melting temperature (Tm) : The temp @ which 50% of the double stranded helical structure is lost –Results: Hyperchromicity (A260) –Applications: Southern blot PCR Loss of viscosity Reem M. Sallam, MD.PhD.

5. Labeled material to detect a target. For DNA: 20-30 nucleotides, complementary to a region in the gene Methods of labeling: IV- Probes: Non-radioactive e.g. Biotin Radioactive e.g. 32 P Sensitive Relatively cheap Hazardous You should follow the radioactive waste disposal regulations. Sensitive Relatively expensive Target DNA Probe Biotin Avidin * Target DNA Probe * Reem M. Sallam, MD.PhD.

6. V- Hybridization: Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

7. VI- Southern Blotting: 1- DNA extraction 2- DNA cleavage (RE) 3- DNA Electrophoresi s (based on size) - + 4- DNA Transfer, blocking, Denature 5- Hybridization e.g. with 32 P- labeled probe 6- Detection Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

8. - - - - - - - - Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005 In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge  they migrate toward the anode. Reem M. Sallam, MD.PhD.

9. Relative mobility Log Mass The electrophoretic mobility is inversely proportional to the log of their mass Biochemistry, 5 th edition, W.H. Freeman and Company, N, 2002, with modification Reem M. Sallam, MD.PhD.

10. http://www.123genomics.com/files/learning.html Example of DNA GEL ELECTROPHORESIS Reem M. Sallam, MD.PhD.

11. DNA Cloning –The amplification of DNA molecule (production of many copies) In replicating cell In Tubes & thermocycler: polymerase chain reaction (PCR) Reem M. Sallam, MD.PhD.

12. Vectors Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

13. 2 3 1 Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

14. Synthesis of cDNA from mRNA using reverse transcriptase Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

15. Polymerase Chain Reaction PCR Reem M. Sallam, MD.PhD.

16. PCR Amplification cycle: @ 95  C @ 72  C @ 55  C 25-35 times 10 6 -10 9 fold amplification Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

17. Advantages of PCR: Efficiency: millions of copies in few hours Sensitivity: can detect & amplify the target even if it is a millionth of the initial sample Adaptable: Can be used with DNA from any source Technically easy Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005

18. Reem M. Sallam, MD.PhD. Lippincott’s Illustrated Reviews, Biochemistry, 3 rd edition, R Harvey & P Champe, 2005