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

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

3. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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

4. Cloning Vectors VectorMaximum Insert size Approx. No. of clones required in library plasmid10 kb10 x 10 5 lambda20 kb5 x 10 5 cosmid45 kb2 x 10 5 YAC1 Mb10 4 BAC> 500 kb5 x 10 4

5. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 5 Plasmids

6. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 9 Cutting DNA with a restriction enzyme

10. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 16 Using a genetic probe to find bacterial cells with a specific recombinant DNA molecule

17. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 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 Type of Stem CellDevelopmental Potential Early EmbryonicTotipotent Blastocyst EmbryonicPluripotent FetalPluripotent UmbilicalMultipotent AdultMultipotent http://learn.genetics.utah.edu/units/stemcells

26. Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh EditionCHAPTER 14 DNA Technologies 26 Possible therapeutic uses of stem cells Treatment of disease Diabetes Parkinsons disease Treatment of spinal injury Culture of differentiated tissues