Genetic Engineering
Recombinant, Cloning,
Transposons, PCR’s, RFLP’s,&
Gel Electrophoresis
Intro
What is a Transposon?
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Jumping Genes
Discovered by Barbara McClintock 1940’s-50’s
Nobel prize in 1983
Two kinds of Transposons
– Insertion Sequences
• A gene which codes for the enzyme transposase.
• Transposase moves genes from one DNA region to another.
May cause mutations if it transposes a gene for regulation.
– Complex transposons
• Long sequences that include extra genes.
Types of
Transposons
What is Junk DNA?
• DNA that does not code for a protein.
• 97% of the human genome
• What is most of our DNA for?
– Regulatory sequences
– Introns
– Polymorphic Regions: noncoding highly variable DNA
regions.
– Repetitive Sequences
• Tandem repeats within a gene: Huntingtons
• Tandem repeats at ends: Telomeres
Where did it come from?
• One hypothesis about the junk is that these chromosomal
regions are trash heaps of defunct genes, sometimes
known as pseudogenes, which have been cast aside and
fragmented during evolution.
• Evidence for a related hypothesis suggests that
the junk represents the accumulated DNA of failed
viruses.
• Yet another hypothesis is that the junk DNA provides a
reservoir of sequences from which potentially
advantageous new genes can emerge. (Junk vs trash)
• As with most things in science, time and technology will
tell…
What are the tools of Genetic
Engineering?
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Gel Electrophoresis
DNA Probe
Polymerase Chain Reaction
Restriction Fragment Length
Polymorphisms
• Complementary DNA
Gel Electrophosesis
• Separation of large molecules of DNA by
size.
• Argose Gel in an electrical field
Gel Animation
The specifics of the Gel
• DNA has a negative charge (PO4 -3). Flows from
the cathode (-) to anode (+). Hint: repels from
negative.
• Can separate DNA, Proteins, or Amino Acids
• Material used in Gel must by cut by restriction
enzymes to migrate through gel.
• Each restriction fragment is a segment of DNA.
IE. If there is one band on the gel the DNA is
uncut. If there are 2 bands the DNA is cut in
2,etc.
• The first lane represents known DNA for
Running
comparison.
Gel
.
So what is the use of the Gel?
• DNA fingerprinting: Who did it?
• Outbreaks: Diseases change over time
(viral and bacterial morphology change)
• Paternity testing: Who’s the Daddy?
• To see if the genetically engineered
product is imbedded.
AP Test Questions: Quiz Yourself
• In any electrophoresis gel,
which fragment of DNA is
larger?
• A) The one nearer the well
• B) The one farther from the
wells
• Why?
• All of the following are
true of electrophoresis
except
• A) it is used to analyze only
DNA
• B) the heavier the fragment the
slower it moves
• C) the fragments of DNA are
neg. charged and migrate to
the positive pole.
• D) a buffer must cover the gel
to allow a current to pass
through the system.
• E) restriction enzymes cut
DNA in only certain sites on
the strand.
So how is DNA cut?
• Restriction Enzymes.
• Discovered in1960’s, a bacterial defense
mechanism against bacteriophages.
• Examples: EcoRI(from E.coli), BamHI, HindIII
• Work by: cutting DNA at recognition
sequences/sites, I.E. GAATTC
• Cuts are staggered leaving sticky ends.
• Cuts are called Restriction Fragments
Restriction Enzyme EcoRI
How is it used here?
Recombinant DNA is made by
pasting sticky ends together with
ligase.
So what makes your Gel
Electrophoresis different from
everyone else's? (identical twins not included)
• RFLP’s: Restriction Fragment Length
Polymorphisms or RFLPs
– Created by DNA treated with restriction
enzymes
• The noncoding Regions of human DNA is
uniquely yours = DNA Fingerprint
• Inherited through Mendalian Genetics
• Uses: Paternity testing & Convictions of
Crime.
How an RFLP is analyzed
Analyze This
• .
What is recombinant DNA?
• DNA taken from two different sources and
pasting them together as one.
• Two types
– Natural
• Occurs during viral transduction, bacterial
transformation, conjugation, and through
transposons
– Artificial
• Genetic engineering/ biotechnology with
restriction enzymes.
Cloning into a plasmid
HHMI
Genetic
Engineering
Ethical
Implications?
How can genes be cloned?
Insulin
• 1.Isolate gene
&
Diabetes
• 2.Insert into Plasmid
• 3.Insert into vector, I.E. competent
bacterium (a bacteria which can take up the plasmid)
• 4.Clone the gene through binary fission
• 5.Harvest successfully cloned bacteria.
• Why? Protein Production, Gene therapy,
Genetic Engineering
How can a specific sequence of
DNA be tagged?
• With a DNA Probe.
– A radioactive label placed on a DNA fragment.
– The single stranded radioactive marker binds to
its complementary sequence.
• Uses:
– Gel Electrophoresis, to ID a specific sequence.
– Cloning, to ensure gene uptake
– To identify inherited genetic defects: Sickle cell,
Tay-Sachs, Huntington's, etc.
How can we read his DNA? or hers?
• PCR Analysis: Polymerase Chain Reaction
– Automated technique which amplifies/copies
DNA. Billions of copies within hours.
– Need: Nucleotides (A,T,C,& G’s), Primers, Test
tube, Taq Polymerase (Extremophile DNA
Polymerase) (( heat speeds up the process))
PCR
PCR
PCR in Action
Problems with PCR
• Contamination: Need a clean room
environment. No dust mites or bacteria
• Small segments of DNA: if its too big the
primer will attach to the wrong thing, an
incorrect DNA sequence would result.
• The nucleotide sequence must be known
in order to create the correct primers.
• Did I mention contamination?
cDNA’s uses to Bioengineering
• Applications
• It can be used for gene discovery or expression
analysis
• Immediate PCR Amplification of known genes
• Verification of genetic mutation
• Comparison of a specific gene between
different tissues
• Analysis of mRNA alternative splicing
• Gene cloning and target sequencing
How did we get cDNA?
cDNA = complementary DNA
(paging Dr. J Winters)
• Problem: Humans have introns, bacteria
don’t.
• Solution: Retroviral reverse transcriptase.
Makes DNA from your cells mRNA =
complete coding sequence, no introns
needed.
AP Test Questions: Quiz Yourself
• What is true of
biotechnology
techniques?
• A) PCR is used to cut DNA
molecules
• B) A DNA Probe consists of a
radioactive single strand of DNA
• C) Restriction Enzymes were
first discovered in bacteriophage
viruses.
• D) EcoR1 is the name for a DNA
probe
• E) All humans contain the same
RFLP’s
• Which enzyme
permanently seals
together DNA
fragments that have
complementary sticky
ends?
• A) DNA Polymerase
• B) Single stranded binding
protein
• C) Reverse Transcriptase
• D) DNA ligase
• E) RNA polymerase
Ethics
• Safety: is your genetically altered food
safe? Can your food alter you?
• Privacy: DNA chips, Healthcare?
• Eugenics: Insertion of genes, changing
your genetics, sterilizing inferior humans,
gene therapy.
Gene
Therapy
And now onto your Clayton
State Lab….
• Please Read the Gel.
DNA Ladder
A
B
Virtual Labs
PHS: II Electrophoresis
1. Review Electrophoresis steps. Take the quiz.
Paraphrase the question and correct answers.
Use the semi-logarithmic paper to draw the
graph with the paper given. This is part of your
grade!!!!
PHS: I Bacterial Transformation
Crops
Mini
Lab
2. Draw the steps of
bacterial
transformation. Take
the quiz. Paraphrase
the question and
correct answers.
3. There are two labs here. Do both. Take notes on
both. Compare and contrast the techniques. Where
would you use each? What are the limitations of each?
Any final thoughts or interesting points? (You must
have at least one.)