Topic 18, Construction and analysis of DNA clones

1. Definitions:
   A. Recombinant DNA: new combinations of DNA molecules that are not found in nature
   B. Restriction enzyme = restriction endonuclease:  enzyme that cleaves DNA
       at a specific nucleotide sequence
       1. Restriction enzymes are bacterial enzymes that "restrict" or prevent viral
           infection because they cleave the invading viral DNA at DNA sequences
           that are not found in that bacterial genome
   C. Vector: carriers of DNA fragments to be cloned
       1. Plasmid vectors, bacteriophage vectors, cosmids, bacterial artificial
           chromosomes (BACs), yeast artificial chromosomes (YACs)
   D. Clone: multiple identical copies of something
   E. Libraries: collections of cloned DNA sequences
   
   
2. Described how plasmid DNA and DNA from an organism to be cloned
   are both cleaved with the same restriction enzyme and the DNAs ligated together,
   etc.
   
   
3. DNA fingerprinting, presented in detail on handout, summarized here
   A. Polymorphisms:  differences between individuals
       1. Morphological polymorphisms
       2. Fingerprint polymorphisms, a type of morphological polymorphisms
       3. Protein polymorphisms
       4. DNA polymorphisms
   B. Restriction fragment length polymorphism (RFLP), variations in the sizes of
       DNA fragments produced by digestion of DNAs from different individuals
       with a restriction enzyme
   C. Steps to carryout RFLP analysis:
       1. Extract DNA
       2. Cut the DNA into precisely defined fragments with a restriction enzyme
       3. Separate the DNA fragments from each other by electrophoresis
       4. Transfer the separated DNA fragments onto a nylon membrane
       5. Hybridize the fragments (which are on a membrane) with a radioactive
           DNA sequence (probe)
       6. Reveal the position(s) of the radioactive probe using X-ray film
   D. Example of RFLP analysis of the gene for sickle cell anemia
   E. Variable number tandem repeats (VNTRs)
   F. Single locus probes (probes which hybridize with one DNA sequence in a genome)
       vs. multilocus probes (probes which hybridize with more than one DNA
       sequence in a genome)
   
   
4. Polymerase chain reaction (PCR)
   A. A cell-free method of amplifying a specific DNA sequence in a cell
   B. Steps in PCR amplification (uses a thermocycler)
       1. Denaturing temperature, (90-90C) is used to denature the DNA
       2. Annealing temperature (50-70 C) is used to hybridize the primers to the template DNA
       3. Extension temperature (70-75 C) extends the primers in the 5' --> 3' direction
           a. Taq polymerase (from bacteria growing in hot springs) is used for this
       4. This cycle is repeated many times to produce large numbers of copies
           of a DNA sequence between the two primers
   
   
5. DNA sequencing using the dideoxy method
   A. Dideoxy ribose lacks the 3'-OH group which is necessary for attachment
       of the next nucleotide during DNA synthesis
   B. Four DNA synthesis reactions are carried out which each contain all necessary
       components for DNA synthesis (DNA polymerase, dATP, dCTP, dGTP, and dTTP, etc.)
       plus a small amount of dideoxynucleotide for one of the bases.   Thus, the normal
       deoxynucleotide plus a small amount of dideoxynucleotide of that nucleotide would be present.
   C. The resultant DNAs are separated by electrophoresis as shown on the handout
       and the sequence of bases read off of the gel as shown
   D. Automated DNA sequencing
       1. A single tube containing all necessary components for DNA synthesis plus small
           amounts of all four dideoxyribonucleotides each labeled with a different colored
           fluorescent molecule is set up and DNA synthesis is allowed to take place
       2. The product is separated out in a single lane on a gel, and the various fluorescent
           dyes are read by the machine giving the nucleotide sequence

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Updated 12/8/99