Transcript Chapter 19 – Molecular Genetic Analysis and Biotechnology

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