Chapter 19 – Molecular Genetic Analysis and Biotechnology

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

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

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

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

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

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

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

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

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

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

Cloning vectors YACs BACs Shuttle vectors 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

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

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

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

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)

Gel Electrophoresis Results

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

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

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

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

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