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 Sticky/cohesive 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 Recombinant plasmid Ampillicin resistance
Β-galactosidase cleaves X-gal and bacteria is blue
Recombinant plasmid
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 BACs Shuttle vectors Yeast artificial chromosomes
Yeast origin of replication, centromere, telomeres
~600kb – 1,000kb
BACs
Bacterial artificial chromosomes
~ kb
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 Annealing Elongation/extension
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 Bioremediation Agriculture
Bacterial production of human insulin, growth hormone
Bioremediation
Bacteria genetically engineered to break down toxic chemicals
Agriculture
Viral/pesticide resistance; increase nutritional value