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Lecture ONE: Foundation Course Genetics Tools of Human Molecular Genetics I Discuss the following: • Genetic engineering • Molecular Genetics • Genetic Screening • Recombinant DNA • Stem cells Biology, Seventh Edition CHAPTER 14 DNA Technologies 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 3 Copyright © 2005 Brooks/Cole — Thomson Learning Cloning Vectors Vector Maximum Insert size Approx. No. of clones required in library plasmid 10 kb 10 x 105 lambda 20 kb 5 x 105 cosmid 45 kb 2 x 105 YAC 1 Mb 104 BAC > 500 kb 5 x 104 Biology, Seventh Edition CHAPTER 14 DNA Technologies Plasmids 5 Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh Edition CHAPTER 14 DNA Technologies Cutting DNA with a restriction enzyme 6 Copyright © 2005 Brooks/Cole — Thomson Learning Restriction Enzymes fig 14.2 • 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 14-1) • 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 Biology, Seventh Edition CHAPTER 14 DNA Technologies Cutting DNA with a restriction enzyme 9 Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh Edition CHAPTER 14 DNA Technologies • 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 10 Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh Edition CHAPTER 14 DNA Technologies Producing a genomic or chromosome library 11 Copyright © 2005 Brooks/Cole — Thomson Learning • 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 Biology, Seventh Edition CHAPTER 14 DNA Technologies 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 13 Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh Edition CHAPTER 14 DNA Technologies • Genetic probes • Segments of single-stranded DNA that can hybridize to complementary base sequences in target gene • Southern blot technique 14 Copyright © 2005 Brooks/Cole — Thomson Learning Southern Northern, and Western Blotting •Southern Blotting: DNA •Northern Blotting: RNA •Western Blotting : Protein Biology, Seventh Edition CHAPTER 14 DNA Technologies Using a genetic probe to find bacterial cells with a specific recombinant DNA molecule 16 Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh Edition CHAPTER 14 DNA Technologies • 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 17 Copyright © 2005 Brooks/Cole — Thomson Learning Biology, Seventh Edition CHAPTER 14 DNA Technologies Amplification of DNA by PCR 18 Copyright © 2005 Brooks/Cole — Thomson Learning 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. 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. Gel Electrophoresis • A method of separation pieces of DNA • Horizontal gel (Agarose) • Vertical (Polyacrylamide) • Many variations on the methods exist 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 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 Stem Cells are undifferentiated cells Type of Stem Cell Early Embryonic Blastocyst Embryonic Fetal Umbilical Adult Developmental Potential Totipotent Pluripotent Pluripotent Multipotent Multipotent http://learn.genetics.utah.edu/units/stemcells Biology, Seventh Edition CHAPTER 14 DNA Technologies Possible therapeutic uses of stem cells • Treatment of disease • Diabetes • Parkinsons disease • Treatment of spinal injury • Culture of differentiated tissues 26 Copyright © 2005 Brooks/Cole — Thomson Learning