PCR.html: 20_07PCR.jpg
A DNA sample can be copied many times (amplified) in vitro by the polymerase chain reaction PCR
in a 3-step cycle.
(4) The blot is exposed to a radioactive probe, a single-stranded DNA complementary to the β-globulin gene, which base-pairs (hybridize) to the restriction fragments. _Vid_Johnson4e/2_139-Hybridization.rm | (5) Photographic film is laid over the blot (autoradiography). The radioactivity in the probe exposes the film to form an image corresponding to the fragments. |
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Cloning is used to make many copies of a gene of interest.
A hummingbird gene is inserted into a plasmid (cloning vector) from E. coli
with a restriction enzyme.
The plasmid contains the ampR gene,
which makes E. coli cells resistant to the antibiotic ampicillin.
It also contains the lacZ gene
encoding β-galactosidase;
this enzyme digests the sugar X-gal to form a blue product.
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Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony.
Colonies with nonrecombinant plasmids will be blue,
because they can digest X-gal.
Some of the recombinant clones have a disrupted lacZ gene;
they will form white
colonies.
These white
colonies can be analyzed
to identify those carrying the gene of interest.
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Nuclear Transplantation - Dolly.
The nucleus of a differentiated mammary cell is transplanted to
an enucleated egg cell from a female.
The resulting embryo is implanted in the uterus of a surrogate mother.
The cloned animal is identical in genetic makeup to the donor supplying the nucleus.
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Gel electrophoresis
is used for separating nucleic acids or proteins that differ in size.
The molecules are separated on the basis of their rate of movement through a gel
in an electric field.
How far a DNA molecule travels while the current is on is inversely proportional to its length.
After the current is turned off, a dye is added; this reveals the separated
bands
by fluorescing in ultraviolet light.
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DNA fingerprints from a murder case.
This autoradiograph shows that DNA in blood from the defendant's clothes matches the DNA fingerprint of the victim but differs from that of the defendant.
This is evidence that the blood on the defendant's clothes came from the victim, not the defendant.
The three DNA samples were subjected to Southern blotting using radioactive probes.
PCR
can also be used to amplify the amount of DNA collected at the crime scene.
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Restriction fragment analysis
The normal allele of the ß–globin gene, which produces a subunit for the oxygen-carrying hemoglobin,
has two sites recognized by the Dde I restriction enzyme, which cuts the DNA
into three restriction fragments.
The sickle-cell mutation destroys one of the Dde I restriction sites.
Digestion of mutant DNA with Dde I generates two fragments
rather than the normal three.
These fragments can be identified by gel electrophoresis.
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Plasmids are circular DNA molecules that replicate separately from the bacterial chromosome.
A plasmid from a bacterium can be used to clone a gene of interest from another organism.
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After cloning in a host cell, multiple copies of a gene of interest can be harvested.
The genes are useful for basic research, and their proteins can be applied to many uses.
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Three-stage approach of the Human Genome Project.
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Hybridization with a complementary nucleic acid probe
detects a specific DNA within a mixture of DNA molecules.
Cells containing recombinant plasmids are transferred to a master plate and grow into visible colonies.
Colonies of cells containing the gene of interest can be grown in large tanks of liquid growth medium.
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A genomic library is a collection of many bacterial or phage clones,
each containing copies of a particular DNA segment from a foreign genome.
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Using a restriction enzyme and DNA ligase to make recombinant DNA.
The restriction enzyme in this example (called EcoRI) recognizes a specific sequence, (restriction site) and makes staggered cuts in the sugar–phosphate backbones, producing fragments with sticky ends.
Any fragments with complementary sticky ends can base–pair;
if they come from different DNA molecules, recombinant DNA is the product.
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Using a restriction enzyme and DNA ligase to make recombinant DNA.
The restriction enzyme
in this example (called EcoRI) recognizes a specific sequence (restriction site)
and makes staggered cuts in the sugar–phosphate backbones, producing restriction fragments with sticky ends.
Fragments with complementary sticky ends can base–pair and the gaps sealed with
DNA ligase.
If the fragments come from different DNA molecules,
recombinant DNA is the product.
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Whole-genome shotgun approach to sequencing.
In this approach, developed by Celera Genomics,
random DNA fragments are sequenced and then ordered relative to each other.
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Southern blotting can be used to compare restriction fragments of DNA. | ||||
---|---|---|---|---|
(1) DNA samples are mixed with Dde1 restriction enzyme. | (2) Restriction fragments are separated by gel electrophoresis. | (3) Capillary action pulls alkaline solution up the gel, transferring the DNA to the nitrocellulose paper. The DNA stuck to the "blot" are positioned in bands corresponding to those on the gel. |
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Embryonic stem cells harvested from the blastocyst are
totipotent, able to differentiate into all cell types.
Adult stem cells harvested from tissues such as bone marrow, intestinal wall, and brain,
are pluripotent, able to give rise to a few cell types.
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APPLICATION
The sequencing of nucleotide fragments up to 800 base pairs can be determined by specialized machines that separate the
labled reaction products by length.
TECHNIQUE
Each strand stars with the same primer and ends with a di-deoxyribonucleotide (
ddNTP
).
Incorporation of ddNTP terminates a growing DNA strand because it lacks a 3'-OH to attach the next
nucleotide.
By tagging the ddNTP with different fluorescent dyes, the oroginal nucleotide sequence can be determined.
RESULTS
The color of the fluorescent tag on each strand identifies the nucleotide at its end.
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Some differentiated somatic cells in plants are totipotent.
They can reverse their differentiation and then give rise to all the cell types in a mature plant,
a clone of the original plant.
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EXPERIMENT
Frog egg cells are exposed to UV
light, destroying the nucleus.
Nuclei from cells of embryos are transplanted into the enucleated egg cells.
RESULTS
Most of the recipient eggs developed into tadpoles when the transplanted nuclei
came from relatively undifferentiated cells of an early embryo.
CONCLUSION
The nucleus from a differentiated frog cell can direct development of a tadpole.
However, its ability to do so decreases as the donor cell becomes more differentiated.