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

– 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– Ampillicin resistance– Β-galactosidase cleaves

X-gal and bacteria is blue

• Recombinant plasmid– Ampillicin resistance– 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

– 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

– 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

– Bacterial production of human insulin, growth hormone

• Bioremediation– Bacteria genetically engineered to break down

toxic chemicals

• Agriculture– Viral/pesticide resistance; increase nutritional

value