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DNA Technology
Chapter 20
Plasmid Use
Plasmids are good tools for DNA
Technology
 Can be isolated from bacterial cells
 Often carry resistance genes
 Isolated genes of interest can be inserted
into the plasmid
 How is this insertion done?
 Restriction endonucleases (enzymes)

Restriction Enzymes
Where were restriction enzymes first found?
 Bacterial cells
 They were used to protect bacteria from
intruding phage DNA
 Bacterial DNA is modified to protect it
from its own restriction enzymes
 Restriction enzymes often cut DNA
leaving “sticky ends”

Restriction sites are the regions on the
DNA that the restriction enzyme cuts.
 Why are restriction sites palindromes?

How are
restriction
enzymes
used in DNA
technology?
Cloning Genes of Interest
How can a biologist make large amounts of a
gene and thereby produce lots of protein
products?
 Clone the genes in recombinant plasmids

Which
method of
bacterial
genomic
alteration is
exploited in
this
process?
Genomic
Libraries
cDNA
What is the problem with inserting a human
gene into a bacterial plasmid?
 Introns are not spliced in prokaryotes
 How can this problem be solved?
 Reverse Transcription of mRNA

Why is
cDNA
shorter than
the original
eukaryotic
DNA?
Probes
Radioactive probes can tag a specific gene
sequence within a mass of DNA
 Probes are single stranded compliments
to known sections of the DNA

Gel electrophoresis
How does gel electrophoresis work?
 Uses electric charge to separate
molecules based on their size
 What charge does DNA have?
 Negative
 Which sized fragments will move furthest
through the gel?
 Smallest ones

Restriction Fragment Analysis
Genetic markers are regions of DNA that
vary from person to person
 Usually located on non-coding regions of
the DNA
 Using restriction enzymes and gel
electrophoresis, DNA of different
individuals can be analyzed and compared
 Extract DNA and treat it with restriction
enzymes

The red triangles indicate where the enzyme
cuts the DNA.
Procedure:
The restriction enzyme is added to the
DNA being analyzed and incubated for
several hours, allowing the restriction
enzyme to cut at its recognition sites. The
DNA is then run through a gel, which
separates the DNA fragments according to
size. You can then visualize the size of the
DNA fragments and assess whether or not
the DNA was cut by the enzyme.
Gel with an uncut and cut
samples of DNA. Note that the
sizes of the cut DNA fragments
add up to the size of the uncut
DNA.
How could you detect the differences between
these 2 alleles?
Using RFLP Analysis to Detect
Harmful Alleles
Harmful, disease causing alleles usually
have identical RFLP’s within a family
 Once the known RFLP’s for the normal
and disease causing alleles are known
family members can be tested using
Southern Blot analysis

Southern Blot:
Load the gel with the DNA to be tested.
Markers serve as standards for determining
sizes of DNA fragments.
Separated DNA’s are denatured while still
in the agarose, by soaking the gel in a
basic solution
 Single stranded DNA’s are transferred to
a nylon membrane by blotting

A Radioactively labeled
probe is added to the
nylon membrane
 The probe is either
RNA or DNA that will
compliment a specific bp
sequence on the DNA
 After unbound probes
have been washed away
only bound probes
remain on the blot

VNTR
A VNTR is variable numbered tandem
repeat
 Tandem repeats are interspersed
throughout the genome
 Different VNTR’s can be detected using
southern blot technique

One VNTR is inherited from each
parent
Southern blot analysis usually shows 2
different bands one inherited from each
parent
 How could an individual have one band for
the VNTR?
 He/She inherited the same sized VNTR
from each parent

Three different alleles for this
particular VNTR
What are the
different
genotypes for
these 6
individuals?
Frequency of VNTR’s





Frequency of allele pattern at a single VNTR has
been established for specific sites within the
genome
What is the probability of matching a 5 locus DNA
profile, where each locus is:0.01, 0.02, 0.03, 0.06
and 0.10?
One in 27.8 million people will randomly match
this profile
OJ’s profile was of 24 different loci and he
matched all 24!
The odds were 1 in 10 billion
Amplification of DNA







What enzymes would be necessary for DNA
amplification?
DNA polymerase and ligase
What else would be necessary for the process to
work?
Primers and nucleotides!
How was the original DNA initially uncoiled and
unwound?
Heat
Why did the heat cause difficulty with the
procedure?
Mapping the Entire Genome
Gene linkage mapping uses recombination
frequencies to construct linkage maps of
chromosome
 Chromosome walking will identify
sequential regions of the chromosome

Chromosome
Walking
DNA Sequencing uses defective
nucleotides to sequence the DNA.

In Situ Hybridization
Denatured DNA is placed on slide
 radioactive single stranded probe is used
to identify complementary DNA
 Used to identify genes that different
species have in common

Microassays
use in situ
hybridization
technique to
determine
which genes
are actively
being
expressed in
the tissue
sample
Other uses for the new
technology….
Gene
Therapy
Stem cells are
the best
candidates for
this therapy
Pharmaceutical Products
 Forensics
 Environment
 Agriculture
 Paleontology

Determining Paternity
Which child cannot belong to this set of parents?
Which lane represents the father?
3
Rape
Investigation
Did the suspect
commit the crime?
Supplemental Lab 6A
In this lab we will transform E. coli
bacterial colonies with recombinant
plasmid DNA
 It will be your job to distinguish between
bacterial colonies that have been
transformed with the recombinant
plasmids

Procedure for the Transformation

Click here to find out more about the
procedure
Predict results for your
procedure!