chapter 20

Bio1151 Chapter 20 9

DNA cloning (making copies of a DNA segment) often makes use of a bacterial .
- DNA cloning.
  A plasmid from a bacterium can be used to clone a gene of interest from another organism.
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- Many bacteria contain plasmids.
  Plasmids are circular DNA molecules that replicate separately from the bacterial chromosome.
- After cloning in a host cell, multiple copies of a gene of interest can be harvested.
  The cloned genes are useful for basic research, and their proteins can be applied to many uses.
- Pest resistance in plants.
- Cleaning up toxic waste.
- Proteins that dissolve blood clots.
- Human growth hormone (HGH) treats stunted growth.
Bacterial enzymes can be used to make DNA which can be cloned in host bacteria.
- Restriction enzymes.
  A bacterial restriction enzyme is an endonuclease that recognizes a specific DNA sequence (restriction site).
  Many restriction sites are palindromes: the nucleotide sequences are the same in the two antiparallel strands.
  The endonuclease makes cuts in the restriction site that leaves staggered single-stranded ends.
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- Restriction fragments.
  A restriction enzyme (EcoRI in this example) makes staggered cuts at restriction sites of a DNA molecule.
  The resulting fragments have complementary sticky ends.
  If the fragments are from different species, recombinant DNA is produced with the gaps sealed with DNA ligase.
  Review:
- Cloning recombinants.
  A restriction enzyme is used to insert a hummingbird gene into a plasmid, which acts as a cloning vector and placed back into E. coli cells.
  The plasmid genes amp^R (ampicillin resistance) and lacZ are used as genetic markers to screen for E. coli cells containing the recombinant gene of interest.
  Once identified, the recombinant E. coli cells are cloned to make many copies of the gene of interest.
  Tutorial:
- Many bacteria contain plasmids.
  Plasmids are circular DNA molecules that replicate separately from the bacterial chromosome.
are DNA copies made from processed mRNAs which contain only .
- A mature mRNA, which contains only exons, is used as a template to create a single strand of complementary DNA (cDNA) using reverse transcriptase.
  cDNA is then converted to a double-stranded molecule free of introns.
  Review:
The polymerase reaction (PCR) can make copies of a segment of DNA in .
- Polymerase Chain Reaction (PCR).
  A DNA sample can be copied many times (amplified) in vitro in a 3-step cycle.
- Denaturation: Heating the DNA to separate the double-stranded molecule into single strands.
- Annealing: Cool the mixture to allow complementary primers to hydrogen-bond to the single strands.
- Extension: Add DNA polymerase and nucleotides to replicate each strand.
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  Review:
- PCR (continued).
  During each PCR cycle, the target DNA sequence is doubled.
DNA fragments can be separated by electrophoresis and analyzed to identify alleles.
- Gel electrophoresis can separate nucleic acids that differ in size.
  DNA fragments created by restriction enzymes are separated based on their rate of movement through a gel in an electric field.
  How far a DNA molecule travels 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.
  Review:
- Restriction fragment analysis.
  The normal allele of the beta-globin gene, which produces a subunit for hemoglobin, has two sites recognized by the Dde I restriction enzyme, yielding 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, yielding a restriction fragment length polymorphism (RFLP).
  These fragments can be identified by gel electrophoresis.
  Review:
DNA can be used to determine if two samples of DNA are from the same source.
- DNA fingerprint.
  This gel 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.
- How can DNA samples from a crime scene be amplified to have enough DNA for fingerprinting?
- Use PCR. Exercise:
- Polymerase Chain Reaction (PCR).
  A DNA sample can be copied many times (amplified) in vitro in a 3-step cycle.
- Denaturation: Heating the DNA to separate the double-stranded molecule into single strands.
- Annealing: Cool the mixture to allow complementary primers to hydrogen-bond to the single strands.
- Extension: Add DNA polymerase and nucleotides to replicate each strand.
  continue
  Review:
DNA sequencing can be done by the chain termination method.
- Dideoxy chain termination.
  A DNA fragment is denatured to produce a single-stranded template in a PCR reaction with the following:
- DNA primer
- DNA polymerase
- the four different deoxyribonucleotides (dNTPs)
- four different dideoxyribonucleotides (ddNTPs)
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- During primer extension, the DNA polymerase sometimes inserts a ddNTP instead of a dNTP.
  The ddNTP lacks the 3'-OH needed to attach the next nucleotide, terminating the chain.
  Gel electrophoresis separates the new fragments that terminate at the ddNTP.
  By tagging the ddNTP with different fluorescent dyes, the original nucleotide sequence can be determined.
  Sanger sequencing tutorial:
Some somatic cells remain and can generate a whole organism.
- 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.
Mammalian cells may be or .
- Stem cells.
  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) are pluripotent, able to give rise to a few cell types.
One technique for making of animals is nuclear , which has been used to clone mammals such as Dolly and Copy.
- Nuclear transplantation.
  Frog egg cells are exposed to UV light, destroying the nucleus.
  Nuclei from cells of embryos are transplanted into the enucleated egg cells.
  Most of the recipient eggs developed into tadpoles when the transplanted nuclei came from relatively undifferentiated cells of an early embryo.
  However, the success rate decreases as the donor cell becomes more differentiated.
  video
- Cloning Dolly by nuclear transplantation.
  A mammary cell (with its nucleus) from a female is transplanted into an enucleated egg cell from a different female in vitro.
  The resulting embryo is implanted in the uterus of a surrogate mother.
  The cloned "Dolly" is identical in genetic makeup to the donor supplying the nucleus.
  video
Applications of DNA technology: