Lecture ONE: Foundation Course Genetics Tools of Human Molecular Genetics I

1. Lecture ONE: Foundation Course GeneticsTools of Human Molecular Genetics I

2. Discuss the following: • Genetic engineering • Molecular Genetics • Genetic Screening • Recombinant DNA • Stem cells

3. Recombinant DNA methods • Restriction enzymes • Enzymes from bacteria • Used to cut DNA molecules in specific places • cut DNA is placed in a Vector carrier • DNA ligase joins cut pieces of DNA • Transformation: uptake of foreign DNA into cells

5. Plasmids

6. Cutting DNA with a restriction enzyme

7. Restriction enzymes are molecular scissors which cut DNA at specific base sequences.   E.g. HindIII and EcoR1 are restriction enzymes Hind III cuts DNA at the recognition sequence 5’-AAGCTT-3’ EcoR1 cuts DNA at the recognition sequence 5’- GAATTC-3’ Restriction Enzymes fig 14.2

8. Restriction Enzymes: (Fig 14-1) • Many of the sites in DNA which restriction enzymes recognize are PALINDROMIC • Palindromic: means that the sequence reads the same 5’-3’ in both directions

9. Cutting DNA with a restriction enzyme

10. Splicing foreign DNA into a vector • Foreign DNA and plasmid DNA cut with same restriction enzyme • Produces linear molecules with complementary single-stranded ends • Recombinant DNA created by mixing so sticky ends pair • DNA ligase forms covalent bonds, linking the two fragments

11. Producing a genomic or chromosome library

12. Genomic library • Collection of DNA fragments that represent all the DNA in the genome • Chromosome library • All the DNA fragments in that specific chromosome • cDNA library • Produced using reverse transcriptase • Makes DNA copies of mature mRNA

13. Cloning DNA: • The recombinant DNA can be put back into bacteria and the bacteria allowed to grow • This will produce many genetically identical copies of the piece of DNA. This is called cloning • A clone is a genetically identical individual or cell

14. Genetic probes • Segments of single-stranded DNA that can hybridize to complementary base sequences in target gene • Southern blot technique

15. Southern Northern, and Western Blotting • Southern Blotting: DNA • Northern Blotting: RNA • Western Blotting : Protein

16. Using a geneticprobe to find bacterial cellswith a specificrecombinant DNA molecule

17. Amplifying DNA in vitro by PCR • Small amount of double-stranded DNA • DNA precursors • Specific nucleic acid primers • Taq DNA polymerase • DNA is denatured • Primers attach to primer-binding site on each DNA strand • Each strand acts as template for DNA synthesis

18. Amplification of DNA by PCR

19. Types of PCR I RT PCR Reverses transcriptase PCR. DNA is amplified from mRNA. QRT PCR Quantitative Real Time PCR the amplified product is linked to a fluorescent reporter molecule, the fluorescence is measured at each cycle. This allows the amplification to be monitored to optimize the efficiency of amplification.

20. Types of PCR II Multiplex PCR two or more sets of primers are used in the same reaction mix. Nested PCR (also nested RT-PCR) There are two amplifications made: the: The first amplifying a large product which, in the second amplification is used as the template.

21. Gel Electrophoresis • A method of separation pieces of DNA • Horizontal gel (Agarose) • Vertical (Polyacrylamide) • Many variations on the methods exist

23. The DNA is negatively charged and migrates towards the positive pole The smallest fragments move the fastest through the gel and therefore move the furthest The largest fragments move the least By comparing the fragments in the sample with standards of known size the size of the sample fragments can be estimated

24. Links to virtual labs • DNA extraction http://learn.genetics.utah.edu/units/biotech/extraction/ • Making a gel http://learn.genetics.utah.edu/units/biotech/gel

25. Stem Cells are undifferentiated cells

26. Possible therapeutic uses of stem cells • Treatment of disease • Diabetes • Parkinsons disease • Treatment of spinal injury • Culture of differentiated tissues