Announcements

1
Announcements

• 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
  todays lecture
• 5. Problem set 7 available for practice - not
  graded answers posted Monday.

2
Review of Last Lecture

• I. Origins of mutation - 1 spontaneous, 4
  chemical, and 2 environmental
• II. Mechanisms of DNA repair

3
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? Youve learned of 2
ways to introduce mutations in lab organisms
using EMS (an alkylating agent) and using
site-directed 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?
4
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

5
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

Mendels wrinkled Phenotype in Peas Also Caused
by Transposon
6
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 mothers
  egg

7
II. Uses of Recombinant DNA Technology

• 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
8
Cutting with Restriction Enzymes Sticky or
Cohesive Ends
9
Cutting and Pasting (Annealing and Ligation) of
Sticky Ends
Digest with EcoRI
Complementary base-pairing
DNA ligase
10
Agarose Gel stained with Ethidium Bromide,
Visualized By UV
11
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
12
Joining Blunt Ends
13
III. Vectors - EM of Small Plasmid DNA
Plasmids are vectors, molecular tools for
carrying DNA of interest. Other vectors include
bacteriophage, cosmids, etc.
14
pUC18 Plasmid
DNA fragment up to 5 kbp can insert
Origin of replication
15
IV. Cloning with a Plasmid Vector
Recombinant DNA
Transformation
Selection for cells carrying recombinant plasmids
by plating cells on media with antibiotic.
16
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-containi
ng 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?
17
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.

18
Restriction Mapping problems

Note fragments are linearized to start with.
Figure 18.23
19
Restriction Mapping (contd)
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.

20
Learning Check
Linear DNA fragment
What is the restriction map of this cloned DNA
fragment, showing the locations of the
restriction sites and relative distance between
sites?
Marker EcoRI BamHI EcoRI/BamHI
15
10
9
8
8
7
6
6 5 4 2
5