Lecture 5 – Recombinant DNA Techniques and Biotechnology
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What are restriction enzymes?
o Endonucleases that are named after the bacteria they are isolated
from
o They cut DNA at specific palindromic sequences of 4-8 base pairs –
either staggered or blunt end cuts
How do you determine the frequency of a restriction site in a particular
sequence?
o The longer the restriction site, the longer the DNA fragments
produced by the restriction enzyme.
o The length of the fragment is 4^X power on average
o For example, if the restriction site is 6 base pairs long, the length of
the fragments will be 4^6=4096
How do Bacteria fend off foreign DNA?
o The methylate their DNA to tell the difference
What is a restriction map?
o A linear sequence of the sites at which particular enzymes cut
o It can be measured in bp, kbp, or Mbp depending on the sizes of the
fragments
What is Southern Blotting?
o SNOW DROP
o Sample: DNA
o Mechanism:
 1. Single-Stranded (denatured) DNA is added to
Electrophoresis Gel
 2. Transfer from Gel to Nitrocellulose Membrane (using a
weight)
 3. Heat Treatment fixes DNA to the membrane
 4. Incubate with DNA Probes to allow for hybridization
 5. Wash and prepare Autoradiogram to view DNA Fragments
that are complementary to labeled probe
What is a Zoo Blot?
o A Southern Blot that compared the same DNA sequence in different
species.
What is a clinical correlate for Southern Blotting?
o Fingerprinting
What type of mutation causes Sickle Cell Anemia?
o An Adenine is changed to a Thymine in the 6th amino acid of B-globin
How does the mutation cause Sickle Cell Anemia?
o The mutation eliminates the cleavage site for the enzyme MstII, which
means it is not cut at the right place to form the normal protein.
Which protein allele (Sickle Cell or Normal) is heavier? What does this mean
for an Electrophoresis Gel?
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o The Sickle Cell protein is heavier than the Normal because it was not
cleaved.
o This means that the Normal protein will travel faster on the
Electrophoresis Gel, while the Sickle Cell protein will travel slower.
o If there is a heterozygote, the gel will show one heavy line and one
lighter line because the individual has one allele for each phenotype.
What does the enzyme DNase do?
o It cleaves phosphodiester bonds (which cuts DNA)
In the case of Globin, under which circumstances can DNase cut
hypersensitive sites and under which circumstances can it not?
o 14-Day Erythroblasts: DNase CAN cut hypersensitive sites because
this is not methylated
o MSB: DNase CAN NOT cut hypersensitive sites because this IS
methylated
When looking at a Gel Electrophoresis of a Southblot of the 14-Day
Erythroblasts and the MSB (both given DNase), what would you expect to
see?
o The DNase cleaves the 14-Day Erythroblasts so the fragments will be
smaller and travel faster (and maybe even completely off) of the gel.
o The DNase does NOT cleave the MSB, so it should travel slower down
the gel.
What is a Northern Blot?
o SNOW DROP
o A Northern Blot uses the same Technique as the Southern Blot, but
the sample is RNA, not DNA.
o Because the sample is RNA, no restriction enzymes are needed to cut
it because it is not double stranded to begin with.
What are the differences between Northern/Southern Blots and
Microarrays?
o In Southern/Northern Blotting, the DNA/RNA is immobile, and you
wash it with a mobile probe.
o In Microarrays, the probes are fixed to the plate and the cDNA is
mobile and searches for its complement
What is Expression Array Analysis?
o You isolate and compare mRNA from cells at two stages in
development.
o Reverse Transcriptase is used to produce an RNA/DNA Hybrid =
cDNA
o Two different fluorescent proteins are used to mark the two different
stages of DNA
o cDNA is then denatured so that it can bind to its (immobile) probe on
the Microarray
o The color expression determines which cDNA binds to which probe
What is Comparative Genomic Hybridization (CGH)?
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o It compares a normal patient’s DNA to a sick patient’s DNA (cancer
patient) – using a microarray
o Each patient’s DNA is marked with a different fluorescent marker.
The DNA travels to specific probes and the color allows for us to see
which patient’s DNA bound to which specific probes for comparison.
o This allows you to determine which proteins are expressed in cancer
patients versus regular patients.
What do you need to join two complementary staggered ends?
o Ligase + ATP
What do you need to join two complementary blunt ends?
o Ligase + ATP
What do you need to join a blunt end with a staggered end?
o Deoxynucleotides + Polymerase + Ligase + ATP
Describe the process of cloning:
o 1. The Cloning Vector (plasmid) is cut with a restriction enzyme.
o 2.The Eukaryotic Chromosome is cut with the same restriction
enzyme.
o 3. Fragments are ligated to the prepared cloning vector (DNA LIGASE)
o The recombinant vector is introduced into the host cell.
o Cloning of the host cells produces many copies of the recombinant
DNA
pBR322 is a plasmid where foreign DNA was inserted and cloned. Explain
the mechanism and how you can tell if the plasmid took up the foreign DNA.
o 1. The pBR322 plasmid is cleaved at the ampicillin resistance element.
o 2. Foreign DNA is ligated into this area, and the ampicillin-resistance
element is disrupted in those plasmids that took up the foreign DNA.
o 3. Because the tetracycline gene is still intact, the colonies are grown
on agar containing tetracycline. Only cells that have taken up the
plasmid will grow on the plate
o 4. The colonies that grew on the tetracycline are then transferred to
the same marked location on both tetracycline and ampicillin +
tetracycline plates.
o If the host DNA has the plasmid that contains the foreign DNA, then it
will NOT grow on the ampicillin plate (because the gene was
disrupted) but it WILL grow on the tetracycline plate.
Hybridization can be used to identify a clone with a particular DNA segment.
Explain the mechanism.
o Start with an agar plate with transformed bacterial colonies.
o Press nitrocellulose paper onto the agar plate so some cells from each
colony stick to the paper
o Treat with alkali to disrupt cells and expose denatured DNA
o Incubate the paper with a radiolabeled probe, and then wash
o Probe anneals to colonies of interest.
o View them under x-ray film
When you prepare a cDNA library, what type of molecule do you start with?
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o An mRNA (which is single stranded)
How do you get from a single stranded mRNA to a double stranded cDNA?
o You add reverse transcriptase to bind a complementary DNA molecule
to the mRNA.
o You degrade the mRNA side with RNase
o You synthesize the complementary strand with DNA Polymerase (the
rest of the RNA that was not degraded with RNase acts as a primer)
What is the major difference between a Genomic Library and a cDNA
Library?
Genomic Libraries cDNA Libraries
Source of DNA?
Chromosomal DNA mRNA
Enzymes to make
Restriction
RNase/DNA
library?
Endonuclease/ DNA Polymerase/DNA
Ligase
Ligase/Reverse
Transcriptase
Contains introns?
Yes
No
Cloned genes contain
Yes
No
introns?
Promoter/Enhancer
Yes
No
Present?
Gene can be expressed No
Yes
in the host?
Can be used for gene
No
Yes
therapy?
Name the specific vectors that can be used in DNA Cloning and when they are
most useful.
Vector
Plasmid
Bacteriophage
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
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Host
Bacterium
Bacterium
Bacterium
Yeast
Insert Size (kbp)
<15
<23
<300
<2000
Library
cDNA
cDNA
Genomic
Genomic
What type of sequence must be present for a vector if a bacterial host is
needed?
o A Shine-Dalgarno Sequence
If you are sub-cloning a DNA sequence to express a particular protein, where
should you ligate the foreign DNA into the vector to promote the most
successful expression of the protein?
o A strong promoter must be used so that the mRNA that is transcribed
from the foreign DNA that was added is overexpressed.
Human insulin is produced using bacteria. What is the precursor to insulin?
o Proinsulin.
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Proinsulin has two chains. Why is each separate chain coded for in two
different bacteria?
o Bacteria are unable to process human proinsulin, so each chain is
produced separately and then folded using specific disulfide bridges
afterward.
There is a large Shuttle Vector that can be used for propagation of genes in
bacteria that can be expressed in eukaryotic cells. Elements are added for:
Replication, Selection, Expression, and Purification. Which elements are
added for each of these?
o Replication
 Ori Sites (pUC ori)
o Selection
 Ampicillin R
 Neo R
o Expression
 Pcmv (5’ Cap)
 BGH pA (poly A tail)
o Purification
 In the polylinker, you have myc Epitope, 6X His, Term
o Where do you place the foreign DNA in this system?
 In the Polylinker, based on which restriction enzymes you use
to cut.
o When you transform the plasmid into the vector, which resistance
gives you selection?
 Ampicillin
o When you transform the vector into the eukaryote, which resistance
gives you selection?
 Neomycin
How is Green Fluorescent Protein used to see which protein is expressed?
o The GFP is added at particular sits downstream from the regulatory
elements of a specific gene. The expression pattern determines which
regulatory genes code for particular proteins.
What is a transgenic organism?
o A transgenic organism is an animal that has been genetically
engineered in some way.
What are the foreign genes in transgenic organisms called?
o Transgenes
What are the three possibilities that can happen when transgenes are added
to transgenic organisms?
o Gene Replacement – the mutant gene replaced the original gene and
only the mutant gene is active.
o Gene Knockout – both the mutant gene and the original gene are
completely inactivated or deleted, so neither gene works.
o Gene Addition – both the mutant gene and the original gene are active
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When studying various effects on genes, match the transgenic possibility to
the particular study goal.
o Gene Replacement – Point Mutation Study
o Gene Knockout – Gene Deletion Study
o Gene Addition – Inducible Gene Study or Dominant Negative Mutation
If you test a group of different species and see a sequence that has been
strongly conserved throughout the results, what could you conclude?
o That it is probably a regulation sequence.
How can you use the Tet System to show Inducible Gene Expression in mice?
o You add a Tet-Off Gene and a Oncogene to mice.
o Without tetracycline, an enhancer binds, which allows the oncogene
to be transcribed, and the mice die from tumors.
o With tetracycline, the enhancer cannot bind to the oncogene, which
blocks the oncogene from being transcribed and the tumor recedes.
In the Tet System, what is tetracyclin acting as?
o A repressor of the enhancer
When you add a gene modification in mice at an early embryonic stage, the
zygotes will have some mutated copies of the gene and some normal copies.
If you want a mouse with 100% mutated copies of the gene, what do you do?
o You breed the mice with the mutations until you get a homozygote
with the mutation.
What is the term for the mice that have one mutant copy of the gene and one
normal copy of the gene?
o Chimeric Mice
When you are gene targeting for both the Neo and the tk genes, what
phenotype are you trying to get?
o Neo+ tkWhat is the positive selection in the gene targeting?
o G418 resistance via the Neo+
What is the negative selection in the gene targeting?
o The tk-.
o When tk is expressed, it provides a target for the toxic agent
gancylovir
What type of recombination gives you the Neo+ tk-?
o Homologous recombination
What does Nonhomologous Recombination give you?
o Neo+ tk+
In another study, scientists knocked out a lmxlb gene. In doing this, what did
they see?
o The absence of the gene affected the development of dorsal limb
structures in mice. (The paws were mutated)
What is Site-Directed Mutagenesis?
o A complementary sequence is added which is different than the
template by one specific base. It still binds because the sequence is
similar enough. Then the strands are replicated, transcribed, and
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translated which results in some normal protein and some mutated
protein.
When they produced a Site-Directed Mutagenesis on the XPD Gene, what
happened to the mouse?
o It aged prematurely.
What are three methods that can be used to introduce genes into DNA for
gene therapy?
o Viruses
o Lipsomes
o Electroporation
What are RNAi?
o RNA interference
o They are small RNAs derived by Dicer’s
o They can be involved in silencing endogenous genes
o This is similar to the miRNA’s that combine with argonaute to form
the RISC complex and silence genes.
What is the difference between siRNAs and mi RNAs?
o siRNAs are exogenous
o miRNAs are encoded in the genome
How do siRNAs apply to infectious disease in the Herpes Simplex Virus
example?
o Mice are infected with Herpes Simplex Virus
o The addition of particular siRNA’s can increase the survival of the
infected mice.
o Just HSV with no siRNA leads to the lowest percentage of survival.
o The addition of some siRNAs increases the survival more than others.
What is the difference between reproductive cloning and therapeutic
cloning?
o In reproductive cloning, the early embryo is implanting into a foster
mother.
o In therapeutic cloning, the early embryo is transferred to a culture
dish.
What type of embryo had to be used to get Embryonic Stem Cells?
o The Blastocyst
Scientists then found a way to reprogram adult stem cells instead of using a
blastocyst. What is the name of these cells?
o Pluripotent Stem Cells (iPSCs)