Chap 20 Biotechnology

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A DNA sample can be copied many times (amplified) in vitro by the polymerase chain reaction PCR in a 3-step cycle.

  1. Heating to denature the DNA: separate the double-stranded molecule into single strands.
  2. Annealing to allow complementary primers to form by cooling the mixture.
  3. Extension to replicate each strand with a heat-resistant DNA polymerase and nucleotides.
During each PCR cycle, the target DNA sequence is doubled.

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(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. Continue

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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.

  1. Cytogenetic map Chromosome banding pattern and location of specific genes by fluorescence in situ hybridization ( FISH ).
  2. Genetic (linkage) mapping Ordering of genetic markers.
  3. Physical mapping Ordering of fragments cloned in phage and plasmid vectors; this gives the actual distance in base pairs between markers.
  4. DNA sequencing Determination of nucleotide sequence of each small fragment and assembly of the partial sequences into the complete genome sequence.

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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.

  1. Shown are three “books” in a plasmid library. Each “book” is a clone of bacterial cells containing copies of a particular foreign genome fragment in its recombinant plasmid.
  2. These genome segments can also be stored in a phage library.

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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.

  1. Cut the DNA from many copies of an entire chromosome into overlapping fragments short enough for sequencing.
  2. Clone the fragments in plasmid or phage vectors
  3. Sequence each fragment
  4. Order the sequences into one overall sequence with computer software.

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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.