Download dna technology

DNA TECHNOLOGY
D. Jones
Recombinant DNA
• DNA that contain DNA from different
sources
– Occurs naturally
• Transduction
• Bacterial conjugation
• transposons
– Occurs artificially
• Recombinant DNA technology
Transduction
• Genes are transferred between prokaryotes
by viruses
• Virus are packaged with little bits of
prokaryotic DNA and transfer this DNA as
they infect new cells
Bacterial Conjugation
• Genes are transferred directly from
prokaryote to prokaryote
• Can only occur between cells of opposite
mating types
– f factor special piece of DNA codes for ability
to produce pili
Transposons
• Piece of DNA that can move from one place to
another in the genome
– from one place to another on one chromosome
– from one chromosome to another chromosome
• Sometimes is “cut and pasted” to new location
• Other times is “copied and pasted” too another site
• Does not depend on complementarity
Insertion Sequences
• Contain one gene for transposase ( catalyses
transposition)
• Between two inverted repeats of DNA (noncoding
sequences)
– Upside down , backward versions
• Ex ATCCGGT
ACCGGAT
•
TAGGCCA
TGGCCTA
– Targeted by transposase
• DNA Polymerase creates direct repeats at new
site
• Do not help bacteria in any way
Composite Transposons
• Include extra genes beside transposase
– Extra genes are between two insertion
sequences
• Help bacteria adapt to new environments
– Can move genes for antibiotic resistance
Recombinant DNA Technology
Steps In Recombinant DNA
• 1. Use restriction enzymes to cut gene of
interest from DNA
• 2. Place DNA of interest into vector
(plasmid) using restriction enzymes and
ligase
• 3. Place vector into bacterial cell
• 4. Clone bacteria
PGLO Lab
• You were provided with a plasmid that had the
PGLO gene inserted
• You transferred the plasmid to E. coli bacteria by
– Inducing competence
• Treating them with Ca++ (CaCL2)
• Helping absorption with a heat-shock
• You cloned the cells
– Grew them on agar plates in an incubator
• You tested for competence
– Growing the bacteria on the LB/AMP plates
– Growing bacteria on the LB/AMP/ARA plates and
using the UV light
Gel Electrophoresis
• Used to separate nucleic acid or proteins
– Based on size, electrical charge, etc.
• For nucleic acids distance traveled is
inversely proportional to size
• Dyes are added to visualize the DNA
Steps in Gel Electrophoresis
• 1. DNA to be analyzed is digested with a
•
•
•
•
•
restriction enzyme
2. Tracking Dye is added
3. Load samples into wells in agarose gel
4. Apply electrical charge to the gel
5. Stain with Dye
6. Measure distance traveled from well
Gel Electrophoresis Lab
• You used predigested DNA
– From the lambda virus
– Some was cut with the restriction Hind III
– Some was cut with the restriction enzyme
EcoRI
– Tracking dye was already added to the DNA
Lab Continued
• An agarose gel was placed in an electrophoresis
chamber and covered with buffer
• Undigested DNA and two samples of predigested
DNA were added to separate wells in the gel
• The gels were run, stained and measured.
• Data was graphed and the size of the unknown
EcoRI digest were determined from the known
sizes of the HindIII digest.
Result
Polymerase Chain Reaction
(PCR)
• Amplifies (makes many copies) of a segment of
•
•
•
•
•
•
DNA
Fast
Start with DNA containing the “target”nucleotide
sequence.
Add heat resistant DNA polymerase(comes from
bacteria that live in hot springs)
Nucleotides
Primers(short, synthetic singe stranded DNA)
Complementary to the ends of the “targeted DNA”
Double DNA every 5 minutes.
Advantages of PCR
• Quick
• Selective
• Can use very small amounts of DNA
Restriction Fragment Analysis
• Detects differences in nucleotide sequences
• Digest long DNA molecule with restriction
enzymes
• Sort by size using electrophoresis
• Use to identify small DNA molecules of
viruses and plasmids
• Prepare pure samples of DNA fragments
Comparing the Alleles of a Gene
• Cut DNA with restriction enzymes
• Electrophoresis
• Tag with a radioactive single stranded
nucleic acid
• Southern blot
• diagram
Southern Blotting
• Detects and analyzes DNA sequences
• Can use large DNA molecules
• Make DNA bands of interest visible using
radioactive probe (entire genome is smear)
– Probe is single stranded DNA
– Base pairs with gene of interest
• Photographic plate set on top is exposed by
radioactive probe
– Provides picture of bands
RFLP’s
• Recognized by Southern Blotting
• Can be used for linkage mapping
– The more often 2 RFLP markers are inherited
together the closer together they are on a
chromosome
Human Genome Project
• Restriction enzymes, DNA cloning, gel
electrophoresis. Labeled probes and micro
arrays have made it possible to map the
human genome
• Began in 1990
• Projected completion 2003
Goals of the Project
• identify all the approximately 30,000 genes in
•
•
•
•
•
human DNA,
determine the sequences of the 3 billion chemical
base pairs that make up human DNA,
store this information in databases,
improve tools for data analysis,
transfer related technologies to the private sector,
and
address the ethical, legal, and social issues (ELSI)
that may arise from the project.
Where did the DNA Come From?
• In the Human Genome Project (HGP), researchers
collected blood (female) or sperm (male) samples
from a large number of donors. Only a few
samples were processed as DNA resources, and
the source names are protected so neither donors
not scientists know whose DNA is being
sequenced.
• The human genome sequence generated by the
private genomics company Celera was based on
DNA samples collected from five donors who
identified themselves as Hispanic, Asian,
Caucasian, or African
DNA Sequencing
• DNA sequencing, the process of
determining the exact order of the 3 billion
chemical building blocks
How is DNA Sequencing Done?
• Chromosomes must be broken into much shorter
pieces.
• Each short piece is used as a template to generate
a set of fragments that differ in length from each
other by a single base that will be identified in a
later step
• The fragments in a set are separated by gel
electrophoresis New fluorescent dyes allow
separation of all four fragments in a single lane on
the gel.
• The final base at the end of each fragment is
identified. This process recreates the original
sequence of As, Ts, Cs, and Gs for each short
piece generated in the first step.
• Automated sequencers analyze the resulting
electropherograms and the output is a four-color
chromatogram showing peaks that represent each
of the 4 DNA bases.
• After the bases are "read," computers are used to
assemble the short sequences (into long
continuous stretches that are analyzed for errors,
gene-coding regions, and other characteristics.
Cloning
• Embryo cloning:
medical technique
which produces twins
or triplets
– duplicates the process
that nature uses to
produce twins or
triplets
• One or more cells are removed from a fertilized
embryo and encouraged to develop into one or
more duplicate embryos.
• produces organisms with identical DNA
Protoplast fusion
• Combine two cells into one
Dolly