Transcript Document

Goals of today’s lecture
1) Explain how the polymerase chain reaction
(PCR) works
2) Explain the basics of DNA sequencing
3) Explain how one can map human disease
genes
4) Describe some strategies for genetic
engineering in humans and plants
Figure 19-6
PCR primers must be located on either side of the target
sequence, on opposite strands.
5
3
Primer
3
5
Primer
Region of DNA to
be amplified by PCR
When target DNA is single stranded, primers bind and allow
DNA polymerase to work.
5
3
3
5
3
Primer
Primer
5
3
5
Figure 19-7-1
THE POLYMERASE CHAIN REACTION IS A WAY TO
PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE
Primers
5
3
5
3
3
5
3
5
dNTPs
1. Start with a solution
2. Denaturation
containing template DNA,
synthesized primers, and
an abundant supply of
the four dNTPs.
Heating leads to
denaturation of the
double-stranded DNA.
Figure 19-7-2
THE POLYMERASE CHAIN REACTION IS A WAY TO
PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE
5
3
5
5
3
5
5
3
3
5
5
3
3
5
3. Primer annealing
4. Extension
At cooler temperatures,
the primers bind to the
template DNA by
complementary base
pairing.
During incubation, Taq
polymerase uses dNTPs to
synthesize complementary
DNA strand, starting at the
primer.
Figure 19-7-3
THE POLYMERASE CHAIN REACTION IS A WAY TO
PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE
5. Repeat cycle
6. Repeat cycle again,
of three steps (2–4)
again, doubling the
copies of DNA.
up to 20–30 times, to
produce millions of
copies of template DNA.
Figure 19-10-1
FLUORESCENT MARKERS IMPROVE SEQUENCING
EFFICIENCY.
DNA polymerase
1. Do one sequencing reaction instead of four. Reaction
mix contains ddATP, ddTTP, ddGTP, ddCTP with distinct
fluorescent markers. (With radioactive labels, four
reactions are needed—one labeled ddNTP at a time.)
Figure 19-10-2
FLUORESCENT MARKERS IMPROVE SEQUENCING
EFFICIENCY.
Template DNA
2. Fragments of newly synthesized DNA that result
have distinctive labels.
Figure 19-10-3
FLUORESCENT MARKERS IMPROVE SEQUENCING
EFFICIENCY.
Long
fragments
Short
fragments
Capillary
tube
Output
3. Separate fragments via electrophoresis in massproduced, gel-filled capillary tubes. Automated
sequencing machine reads output.
How Was the Huntington’s Disease
Gene Found?
• Huntington's disease is a rare but devastating
genetic illness.
• An analysis of pedigrees from families affected
by the disease suggested that the trait results from
a single, autosomal dominant allele.
• A genetic map (or linkage map) was used to
localize the Huntington's gene relative to other
genetic markers (a gene that has been mapped
previously).
•
Restriction fragment length polymorphisms (RFLPs) are
differences between chromosomes that are measured as the
presence or absence of a restriction endonuclease recognition site.
Figure 19.10 illustrates RFLP analysis.
SOUTHERN BLOTTING
Location of restriction
endonuclease cuts
Sample 1
Double-stranded
DNA
1. Restriction endonucleases cut DNA
sample into fragments of various lengths.
Each type of restriction endonuclease cuts
a specific sequence of DNA.
2. A sample consists of all the
DNA fragments of various
lengths. The sample is loaded into
a gel for electrophoresis.
Figure 19-7 part 1 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc.
Samples from four
individuals
Sample 1
1
2
3
4
Double stranded
DNA
Power
supply
2. A sample consists of all the
DNA fragments of various
lengths. The sample is loaded
into a gel for electrophoresis.
3. Electrophoresis. Use voltage difference to
separate DNA fragments by size. Small
fragments run faster.
Figure 19-7 part 2 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc.
Samples from four
individuals
1
2
3
4
1
2
3
4
Single
stranded
DNA
Double stranded
DNA
Power
supply
3. Electrophoresis. Use voltage
difference to separate DNA fragments
by size. Small fragments run faster.
4. The DNA fragments are
treated to make them single
stranded.
Figure 19-7 part 3 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc.
1
2
3
4
Single stranded
DNA
Stack of
blotting paper
Filter
Gel
Sponge in
alkaline
solution
4. The DNA fragments are treated
to make them single stranded.
5. Blotting. An alkaline solution wicks up into
blotting paper, carrying DNA from gel onto nylon
filter, where it is then permanently bound.
Figure 19-7 part 4 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc.
Labeled DNA probe in
solution in plastic bag
Stack of
blotting paper
Filter
Gel
Sponge in alkaline
solution
5. Blotting. An alkaline solution wicks up
into blotting paper, carrying DNA from gel
onto nylon filter, where it is then
permanently bound.
6. Hybridization with radioactive probe.
Incubate the nylon filter with a solution
containing labeled probe DNA. The
radioactive probe binds to the fragments
containing complementary sequences.
Figure 19-7 part 5 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc.
Labeled DNA probe in
solution in plastic bag
X-ray film
6. Hybridization with radioactive
probe. Incubate the nylon filter with a
solution containing labeled probe
DNA. The radioactive probe binds to
the fragments containing
complementary sequences.
7. Autoradiography. Place filter
against X-ray film. Radioactive DNA
fragments expose film, forming black
bands that indicate location of target
DNA.
Figure 19-7 part 6 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc.
• The Huntington's disease gene was localized to
chromosome 4 by RFLP analysis.
• One gene within the isolated chromosomal region
that was abnormal in people with Huntington's
disease had an unusual number of CAG codons at
the 5' end of the coding region. Healthy individuals
have about 11–25 of these repeats, whereas affected
individuals have 40 or more.
• A genetic test for Huntington's disease uses PCR
to determine the number of CAG repeats.
Figure 19-13-1
USING ENGINEERED VIRUSES TO INTRODUCE
ALLELES INTO HUMAN CELLS
Viral
RNA
Human
RNA
Reverse
transcriptase
1. Engineered retrovirus
2. Recombinant
contains recombinant RNA,
which has
both viral sequences and
human sequences.
genes enter host
cell.
Figure 19-13-2
USING ENGINEERED VIRUSES TO INTRODUCE ALLELES INTO HUMAN CELLS
Double-stranded
DNA version of
Human cell
introduced genes
DNA complementary
to introduced RNA
Reverse
transcriptase
3. Viral enzymes make
double-stranded DNA
version of introduced
genes.
Host
chromosome
4. Recombinant genes
are inserted into host
chromosome and
transcribed.
• Gene therapy has been used to treat a type of severe
combined immunodeficiency (SCID), a fatal genetic
disease whose sufferers have a profoundly weakened
immune system.
•
The type of SCID treated is designated SCID-X1, because it is
caused by mutations in a gene on the X chromosome.
• Within four months after treatment, nine of the
ten boys had normal levels of functioning T cells;
but 30 months later, two had developed a type of
cancer characterized by unchecked growth of T
cells.
• Although gene therapy holds great promise for
the treatment of a wide variety of inherited
diseases, fulfilling that promise is almost certain to
require many years of additional research and
testing, as well as the refinement of legal and
ethical guidelines.
There are currently no plans for using gene therapy to treat
Patients with Huntington’s disease. Why do you think that is?
A) Because the neurons of the brain are difficult to transfect
with transgenes.
B) Because Huntington’s disease is autosomal dominant.
C) Because Huntington’s disease is caused by accumulation of
a mutant protein that aggregates in the neurons.
D) Because there aren’t enough Huntington’s patients to make
the effort profitable.
Chapter 19 Opener Biological Science 2/e ©2005 Pearson Prentice Hall, Inc.
The Agrobacterium
Transformation System
• Agrobacterium tumefaciens is often
used for genetic transformation of
plants through transfer of its Ti
(tumor-inducing) plasmid (Figure
19.16).