Chapter 19 – Molecular Genetic Analysis and Biotechnology

1. Chapter 19 – Molecular Genetic Analysis and Biotechnology

2. Recombinant DNA technology • One molecule composed of two distinct DNA sources • Biotechnology • Development of commercial products; medical applications

3. Restriction endonucleases/ enzymes • Make double-stranded cuts in DNA • Bacterial source – guards against viral invasion • Bacterial DNA is methylated; viral unmethylated • Name of enzymes is an abbreviation of bacterial source • Usually recognizes 4-6 pallindromic sequences

5. Digestion • Blunt ends • Cut both strands of DNA at same location • Sticky/cohesive ends • Produce staggered cuts; single stranded “sticky” ends • Any DNA cut with the same enzyme will have ends with the same sequence • Can combine DNA from different sources and seal cuts with enzyme ligase

6. Gel electrophoresis • Porous gel made of agarose or polyacrylamide • Sample DNA mixed with loading dye that allows for visualization and increases density • Negatively-charged DNA runs toward positive pole when electrical current passes through the gel • Separates fragments based on size • Smaller fragments migrate the furthest – bottom of the gel • Ladder or marker contains fragments of known sizes to aid in determination of sample fragment size • Expose gel to dye • Methylene bue – light box • Ethidium bromide – UV light

7. Southern blotting - DNA • Restriction digestion of genomic DNA and separated by gel electrophoresis • Large number of band sizes produce smear on gel • Fragments are denatured into single-strands and transferred from gel to a thin nylon or nitrocellulose membrane

8. Southern blotting con’t • Membrane is exposed to probe that has been radioactively- or fluorescently labeled • Probe has complementary sequence to target sequence • Unbound probe is rinsed away and bound probe is detected • Northern blotting – RNA • Western blotting - protein

9. Cloning genes • Produces duplicate copies of specific genes • Provides large number of copies • Insert gene of interest into bacterial cells for rapid replication

10. Cloning vector • DNA gene of interest is inserted into a cloning vector • Requirements: • Origin of replication • Unique restriction site • Has only one recognition site • Selectable marker • Antibiotic resistance

11. LacZ • Intact plasmid • Ampillicin resistance • Β-galactosidase cleaves X-gal and bacteria is blue • Recombinant plasmid • Ampillicin resistance • Inserted sequence disrupts β-galactosidase gene; bacteria remains white

12. Expression vectors • Used not just for copies of gene, but to make gene product • Gene expression • Requires sequences for transcription/ translation

13. Cloning vectors • YACs • Yeast artificial chromosomes • Yeast origin of replication, centromere, telomeres • ~600kb – 1,000kb • BACs • Bacterial artificial chromosomes • ~100-500kb • Shuttle vectors • Can be transferred between two different species (bacteria and yeast) • Origin of replication and markers must be recognized by both organisms

14. Polymerase Chain Reaction (PCR) • Amplifies DNA fragments in vitro • Taq polymerase • Isolated from hot spring bacteria Thermus aquaticus • stable at near boiling temperatures • Automated thermocyclers • Computer aided machine that rapidly changes temperature

15. PCR needed components • Target DNA • Primers – 2 different (one for each strand) • Complementary to end sequences • dNTPs • Buffer/Mg ions • Polymerase

16. PCR steps • Denaturation • Separates DNA into single strands • ~90°C • Annealing • Primers complementary pair to DNA strands • ~55°C • Elongation/extension • Polymerase adds new nucleotides to primers’ 3′ end • ~72°C

17. PCR con’t • Produces billions of copies of target DNA in a few hours • Reverse transcription PCR • Makes cDNA from RNA template • Real-time PCR • Quantifies amount after each cycle • Allows measurement of mRNA; amount of gene expression • Limitations • Need to know DNA sequence – at least the ends • Contamination gets amplified as well • Taq polymerase has no proofreading capabilities • Newer polymerases do • Limited to small sizes (less than 2,000kb)

18. Gel Electrophoresis Results

19. Restriction Fragment Length Polymorphisms (RFLPs) • Variation from individual to individual • Helps with linkage studies for gene mapping • DNA fingerprinting • Also uses microsatellites – short tandem repeats • Size of fragment depends on number of repeats

20. DNA sequencing • Dideoxy sequencing • Normal nucleotides dNTPs – deoxyribonucleoside triphosphate • ddNTPs – dideoxyribonucleoside triphosphate • Missing the oxygen at the 3′ carbon • No nucleotide can be added to strand

21. DNA sequencing con’t • 4 reaction tubes are set up – one for each base • DNA is then denatured and run on a gel

22. DNA sequencing con’t • Sequence on gel is complementary to original strand • Automated sequencers use ddNTPs labeled with fluorescent dye • Sample is analyzed by a computer and sequence is graphed out

24. Applications • Pharmaceuticals • Bacterial production of human insulin, growth hormone • Bioremediation • Bacteria genetically engineered to break down toxic chemicals • Agriculture • Viral/pesticide resistance; increase nutritional value