Announcements
1. Lab reports (X-linked cross) due today - start of lecture
2. Pick up lab overview 12 - read and answer pre-lab
questions, due at start of lab (instead of quiz!).
3. On 3x5 card - write 1-2 specific topics you would like
“reviewed” before the exam or questions you have
4. Exam 3 will cover material through end of today’s lecture
5. Problem set 7 available for practice - not graded; answers
posted Monday.
Review of Last Lecture
I. Origins of mutation - 1 spontaneous, 4 chemical,
and 2 environmental
II. Mechanisms of DNA repair
Learning Check
Although mutations are generally considered deleterious
and to be avoided in the “real world”, they are sometimes
intentionally introduced into organisms in research
laboratories.
(1) Why?
You’ve learned of 2 ways to introduce mutations in lab
organisms: using EMS (an alkylating agent) and using sitedirected mutagenesis.
(2) If you are studying a biological process in which no specific
genes have yet been identified, which of the above tools could you use
and what might you learn?
(3) If you are studying a process in which a specific gene is
known to be involved, which tool might you use and what specific
information could you acquire?
Outline of Lecture 30
I. Transposable elements
II. Recombinant DNA - restriction enzymes
III. Vectors
IV. Cloning: using restriction enzymes and
vectors together
V. Practice problems
I. Transposable Elements
• Also called Transposons or “Jumping Genes”; can move
within the genome.
• Present in all organisms; well-studied in bacteria, maize,
flies.
• Discovered in Maize
Mendel’s wrinkled Phenotype
in Peas Also Caused by
Transposon
Transposons in Humans
• Alu family of short interspersed elements (SINEs)
– Moderately repetitive DNA
– 500,000 copies of 200-300 bp repeats
• Medical example: in a male child with hemophilia, a
transposon (LINE) jumped into the gene on X
chromosome responsible for hemophilia
– Not present on either X chromosome of mother
– Present on chromosome 22 of mother
– This mobile element may have “moved” from chr. 22
to X chr. in the precursor cells of the mother’s egg
II. Uses of Recombinant DNA
Technology
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Basic biomedical science
Basic ecological/evolutionary biology
Applied microbiology
Plant genetic engineering
Transgenic animals
Human Genome Project
Medical biotechnology
Forensic science
Recombinant DNA refers to a new combination of DNA
molecules not found together naturally
Cutting with Restriction Enzymes:
“Sticky” or Cohesive Ends
Cutting and Pasting (Annealing and
Ligation) of Sticky Ends
Digest
with
EcoRI
Complementary
base-pairing
DNA ligase
Agarose Gel stained with Ethidium
Bromide, Visualized By UV
MW plasmid
Nicked Circular - May be higher or lower
Linear Accurate
Supercoiled More compact,
so runs faster
Accurate Gel Mobility of DNA
fragments Depends on
Complete Cutting
Joining Blunt Ends
III. Vectors - EM of Small Plasmid DNA
Plasmids are vectors, molecular tools for carrying DNA
of interest. Other vectors include bacteriophage,
cosmids, etc.
pUC18 Plasmid
DNA fragment up to 5 kbp
can insert
Origin of replication
IV. Cloning with a Plasmid Vector
Recombinant DNA
Transformation
Selection for cells carrying recombinant plasmids by plating
cells on media with antibiotic.
Learning Check
When you are doing a transformation in lab, you might add
your recombinant DNA to competent E. coli, incubate on ice
to allow DNA to adhere to the cell wall, heatshock, let cells
recover in liquid media, and plate out on antibiotic-containing
media.
List all of the controls that are necessary in order for you to
interpret your results the next day.
ie. if no cells/colonies grow on your plates, what will you
conclude? How will you determine what part of the expt. went
wrong?
Insertion into a Plasmid can be
Detected by Disruption of -gal
• Only bacteria which have taken
up plasmid grow on ampicillin.
• Blue-white selection:
– white colonies have insert
– blue colonies have no insert
• To see blue color, add IPTG (an
inducer of -galactosidase
expression) and Xgal substrate.
Restriction Mapping problems
*Note: fragments are
linearized to start with.
*
Figure 18.23
Restriction Mapping (cont’d)
Model 1
Model 2
• Same logic can be used on a circular DNA in homework.
Analyze each lane from left to right and any other information
given. Redraw the plasmid each time for each step you solve.
• Make alternative hypotheses and test them against the data.
• Check that total # of fragments = total size of plasmid. There
could be two same-sized fragments in one gel band.
Learning Check
Linear DNA fragment
Marker
EcoRI
BamHI
EcoRI/BamHI
15
10
9
8
6
7
5
8
6
5
4
2
What is the
restriction map of
this cloned DNA
fragment, showing
the locations of the
restriction sites and
relative distance
between sites?