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Exploring Genes
Recombinant Technololgy
Restriction Enzymes
 What
are restriction enzymes and how
are they used?
 enzymes
that recognize specific base
sequences in DNA and cleave the DNA at
those sequences
 cuts DNA into specific fragments for
analysis
Restriction Enzymes

What characteristic do the cleavage sites of
restriction enzymes share?

palindromic
Restriction Enzymes

How do we identify
a particular
restriction enzyme?



based upon host
organism from which
enzyme was isolated
first letter genus
second two letters
species
Restriction Enzymes

What is a restriction
digest and how is it
analyzed?


DNA cut by one or
more restriction
enzymes
gel electrophoresis
Restriction Enzymes

What is Southern blotting and how is it used?

separating mixture of restriction fragments of DNA
by electrophoresis and probing with labeled DNA
Restriction Enzymes
 What
are Northern blots?
 separation
of RNA fragments and probing
with labeled DNA
 What
are Western blots?
 separation
of proteins and probing with
labeled antibody
DNA Sequencing

How is controlled termination of DNA
replication used to determine the sequence of
a DNA molecule?
DNA Sequencing
DNA Sequencing
 How
large are complete genomes?
 X174
virus – 5386 base pairs (bp)
 human mitocondrial DNA – 16, 569 bp
 Haemophilus influenzae – 1,830,137 bp
 C. elegans – 100,000,000 bp
 H. sapiens – 3,000,000,000 bp
DNA Synthesis

How can DNA be synthesized in the lab?

solid-phase synthesis by phosphite triester
method
DNA Synthesis

What does the
activated monomer
look like?
DNA Synthesis
 Why
is the ability to synthesize DNA
chains valuable?
 making
radioactive or fluorescent probes
helps to locate presence of genes
 synthetic probe can be used as primer
 can synthesize customized genes
Polymerase Chain Reaction

What is PCR?


Why is this a valuable tool?


technique used to make many copies of a specific
DNA sequence
amplify a small amount of DNA
What materials are needed?



primers
all dNTP’s
heat-stable DNA polymerase
Polymerase Chain Reaction
Polymerase Chain Reaction
 What
are some of the ways PCR has
been used
 forensics
 paternity
suits
 detection of HIV
 cancer detection
 detection of tuberculosis bacillus
Recombinant DNA
 What
is recombinant DNA?
 novel
DNA molecules
 What
is needed to make recombinant
DNA?
 vector
plasmid
 virus

Recombinant DNA
 What
else?
 restriction
enzyme
 DNA ligase
Recombinant DNA
Recombinant DNA

Any DNA molecule can be made to contain
sticky or cohesive ends
Cloning Vectors

Plasmids and
bacteriophages
serve as useful
cloning vectors

example – pBR322
Cloning Vectors

Lambda phage can destroy host cell or
become incorporated into host cell.
Cloning Vectors

How are phages
used as vectors?
 What advantages
do these modified
viruses have over
plasmids?
Cloning Vectors
 What
are some of the advantages of
using M13 phage as a cloning vector?
 exists
as a single-stranded molecule
 doesn’t kill bacterial host
 can grow large quantities of M13
 can sequence foreign DNA cloned into
M13 with dideoxy method
Cloning Vectors

How is M13 used for
cloning?
Recombinant DNA

What is a genomic
library and how is it
prepared?

collection of genomic
DNA fragments
inserted into a
cloning vector
Recombinant DNA

How does one screen a genomic library to
find which phages contain a particular gene?

grow phage on lawn of bacteria




plaques form where phage infect cells
prepare replica on nitrocellulose sheet
treat with NaOH to lyse bacteria and denature
DNA
hybridize with 32P labeled probe

audoradiography
Recombinant DNA

What are cosmids?

combinations of plasmids and lambda phages


can hold 45 kb inserts
What are bacterial artificial and yeast artificial
chromosomes (BAC &YAC)?

pieces of DNA containing centromere,
autonomous replication sequence (ARS), and
telomeres

can hold 100-1000 kb inserts
Recombinant DNA

What is chromosome walking and how is it
used?

technique of sub-cloning and re-screening used to
analyze long stretches of DNA
Recombinant DNA

What is c-DNA?


DNA made from m-RNA
How is it made?
Recombinant DNA
 How
is c-DNA used?
 placed
in vectors, inserted into bacteria,
forms a c-DNA library
 How
does a c-DNA library differ from a
genomic DNA library?
 c-DNA
contains no introns
Recombinant DNA

How are c-DNA
clones screened?
Recombinant DNA

For what purpose
are gene chips
used?

to measure the level
of gene expression
in eukaryotic cells
via fluorescence
Recombinant DNA

Why must many eukaryotic genes be
expressed in eukaryotic cells?


require posttranslational modification
What is the most effective way of introducing
eukaryotic DNA into host cells?



retroviruses – Moloney murine leukemia virus
Baculovirus – insect cells
Vaccinia virus
Recombinant DNA

What are transgenic
mice?

mice containing and
expressing foreign
DNA
Recombinant DNA

What led to the formation of this oversized mouse?
Recombinant DNA

What is homologous recombination and how
has it been used to cause gene knockouts?
Recombinant DNA

How can foreign
genes be inserted
into plant cells?


Ti plasmids
effective in dicots
and a few monocots
Recombinant DNA

How can foreign
genes be inserted
into plant cells?



electroporation
cereal monocots and
dicots
“gene guns”


DNA coated onto
tungsten pellets
fired at cells
Recombinant DNA
 How
can one construct a new gene by
producing deletions?
 cut
plasmid at two sites with restriction
enzyme and ligate to form smaller plasmid
 cut plasmid at one site, use endonuclease
to remove additional bases, and ligate
Recombinant DNA
 What
is oligonucleotide-directed
mutagenesis?
 used
to replace code for particular amino
acid
 prepare oligonucleotide primer containing a
mutant code for amino acid to be changed
 separate plasmid strands, anneal primer,
allow for replication
 some progeny will contain mutation
Recombinant DNA

What is cassette
mutagenesis?

type of insertion
Recombinant DNA

What are some of the benefits of this
technology?


understanding protein structure, mechanisms of
catalysis, etc.
produce large amounts of useful proteins






insulin
growth hormone
drugs
diagnostic reagents
gene therapy
agriculture