BIOTECHNOLOGY AND BIOMEDICAL TUTORIAL-PCR, RFLP Analysis & Gene Therapy

The Polymerase Chain Reaction (PCR) Can Make Millions of Copies of DNA in a Short Time

• The polymerase chain reaction (PCR) is a rapid way of amplifying (duplicating) specific DNA sequences
• Method was devised by Kary Mullis of Cetus Corporation, Emeryville
  • He recieved a $20,000 bonus and later a Nobel Prize
  • Later the patent was sold to Hoffman-LaRoche for $300,000,000
• DNA heated to high temperature is not destroyed; separates into single strands, but reforms helix when cooled
• PCR Method:
  • DNA to be amplified is put into solution containing:
    • Short DNA “primers” which can bind to the 3′ ends of the DNA
    • The 4 nucleotide bases: A, C, G, T
    • DNA polymerase
      • Special DNA polymerase (Taq polymerase) working at very high temperature is used; isolated from algae living in hot springs
  • DNA is heated to 95 deg C -> single chains
  • Solution is then cooled to 55 deg C; at 55 deg primers bind to ends of the single strand DNA
  • The temperature is now raised to 72 deg and the DNA polymerase causes the synthesis of new complementary strands to all the single strands
  • This is the end of the first cycle
  • The temperature is cycled from 95 to 55 to 72 over and over
  • The DNA is doubled at each cycle and at the end of 32 cycles it has been amplified 1 billion times
  • A cycle can be done in as little as 17 seconds, so it is possible to get a billion-fold amplification in an hour or less including set-up time

  

• The figures shows 2 cycles of PCR
• PCR can be used to dupicate single DNA molecules
• It has been used to amplify DNA samples from extinct species

Cloned DNA in Colonies Can be Identified by Treating a Replica with Complementary DNA Probes

• Often it is desirable to find bacterial colonies that have specific cloned DNA fragments, a specific gene for example
• If some of the DNA sequence is known a short single strand radioactive probe can be made to search for the desired sequence
  • Most DNA probes have 15-50 bases
• A mirror-image replica of the agar plate with the bacterial colonies is made
  • A piece of nitrocellulose paper is placed over the agar plate and pressed down
  • Some of the bacteria are transferred to the nitrocellulose
• The replica is treated with alkali to break up the cells and is exposed to the probe
• The probe binds to DNA having a complementary sequence, labelling those colonies that have it
• Once the colonies having the desired gene have been identified the investigator can go back to the original plate and get living samples to culture

If a Gene is Expressed Antibodies Can be Used to Identify Proteins

• Under certain conditions bacteria can express the protein coded by a cloned gene
  • Bacteria cannot express genomic DNA because it has introns
  • Can express complementary DNA, because it is made from messenger RNA (introns removed)
  • Genomic DNA can be expressed in eukaryotic cells, such as yeast, because they know how to handle introns
• If a protein has been isolated antibodies can be made
• The antibodies can be used to probe a replica plate to find colonies making the protein
• This allows the gene for the protein to be isolated

Blotting Techniques Help to Analyze Restriction Fragments

• Electrophoresis gels can also be analyzed with DNA probes by a Southern blot
• After gel is run a replica is made by blotting the DNA onto a nitrocellulose or nylon filter
• The replica is then treated with probes that reveal specific bands
• Example of a Southern blot:
  • Suppose you are interested in a gene which has 3 restriction sites for a certain enzyme (marked by X in the picture, sample A); one of the sites occassionally mutates and disappears (sample B, from another person)
  • You have a probe which binds at the spot shown
  • You extract the DNA from your cells and cut it with the restriction enzyme
  • This produces hundreds of restriction fragments because the enzyme cuts sites in other genes as well as the one you are interested in
  • If you do a Southern blot and then stain the DNA with a general stain such as ethidium bromide you will see a smear like this because the hundreds of bands run togetherIf you treat the replica with the probe only the bands that bind the probe will light up
  • • The banding shows that when the mutation occurs the 15.2 kilobase band (sample A) disappears and is replaced by a 20.6 kilobase band (sample B)
    • Why doesn’t the 5.4 kilobase band show in sample A? Look at the diagram above to see where the probe binds.
  • The Southern blot is named for the man who developed the technique
  • Other types of blots have been developed:
    • Northern blot: probes RNA
    • Western blot: probes protein, using antibodies

  RFLPs: Different Individuals May Have Different Sizes of Restriction Fragments

  • RFLP = restriction fragment length polymorphism
  • A fancy way of saying, when you cut the genes up (with restriction enzymes) different people have different sized chunks
    • The A & B samples that we discussed Southern blotting are examples: the same enzyme gives a 20.6 kb fragment in one case, and a 15.2 plus a 5.4 kb fragment in the other case
  • To illustrate RFLPs further we will discuss 2 real cases, both associated with the sickle cell anemia mutation
  • Case 1: HpaI sites flanking the globin gene
    • If you take the DNA from 2 different individuals and compare the sequences in regions that do not code for genes you will find differences in 0.2 to 1% of the bases
    • Most of these differences are neutral since they do not affect coding for genes; they tend to accumulate because they are not eliminated by natural selection
    • Sometimes these variations in bases can be used as markers for genes
      • The variation must affect a restriction site
      • It must be very close to a gene
    • In 1978 Kan & Dozy (UC San Francisco) reported that restriction sites of the enzyme HpaI were associated with sickle cell anemia; they hoped the polymorphism could be used for diagnosis
    • The HpaI sites are in non-coding regions flanking the globin gene
    • It was found that the HpaI site downstream from the gene was different in people with normal hemoglobin A and those with hemoglobin S
      • American blacks (about 60%) with hemoglobin S had a 13 kb restriction fragment
      • Those with normal hemoglobin A had a 7.6 kb restriction fragment
    • Probable origin of the association between the 2 genes:
      • Step 1: a mutation occurs producing a 13 kb restriction fragment in people with normal hemoglobin (probably in the Upper Volta region of Africa)
      • Step 2: the sickle mutation occurs in a person with the 13 kb restriction fragment and spreads throughout West Africa & Mediteranean
    • In other parts of the world the sickle mutation apparently occured in a person or several persons with the 7.6 kb restriction fragment (India, Saudi Arabia, East Africa)
    • This RFLP is of limited value for diagnosis because in some parts of the world sickle cell anemia is associated with the 13 kb fragment, while in others it is associated with the 7.6 kb fragment

    Case 2: MstII site in the region with the sickle point mutation

    • The codon region near the sickle mutation is cut by several restriction enzymes
    • The most useful of these enzymes is MstII, which cuts in 3 places near the mutation site:

    The sickle mutation destroys the middle MstII site

    • N in the MstII site code stands for any nucleotide (it can be G, for instance)
    • Hemoglobin A has an MstII site in codons 5 to 7, but changing an A to a T destroys the site in hemoglobin S
    • In normal hemoglobin MstII will produce a fragment of 1.15 kb in this region
    • When the site is mutated the fragment enlarges to 1.35 kb
    • If you do a Southern blot test, this is the pattern you will see for people with the different types of hemoglobin:
    • The AS individual has one gene of each type and this gives him 2 bands
    • Since the restriction site and mutation site overlap this test will give the correct diagnosis 100% of the time (excluding experimental error)

    Gene Therapy Tries to Replace Damaged Genes

    • Cloning techniqes could be used to replace damaged genes with good ones
    • Genes have already been inserted into animal eggs to make transgenic study animals
      • Sickle cell anemia gene has been put into mice, for example
    • Gene insertion into the germline (eggs):
      • Can be used to prevent appearance of a disease
      • Fertilized egg removed from animal
      • DNA inserted into nucleus with micropipette
      • Egg put back into female for development
      • • Technical problems
          • Gene must insert into egg DNA; insertion is random and often fails
          • Gene needs control elements such as promoters- these may be missing in random insertion
          • Attempts to target genes
        • Ethical problems
          • Many object to directly controlling heredity in this way
      • Gene inserted into adult animal
        • This is is an attempt to cure a disease that is already present
        • Usually gene is put into a vector, such as a harmless virus, that can carry the gene into cells
        • Very difficult because millions of cells must receive the gene

      For More Information

      Michael King of the Terre Haute Center for Medical Education has a biochemistry course with an excellent section on Molecular Tools of Medicine. Another online treatment of DNA technology is the MIT Biology Hypertextbook. The Cold Spring Harbor Marine Biology Lab has a DNA Learning Center with animations of PCR and Southern Blotting. The use of restriction fragments in criminal investigation is illustrated in a DNA Detective section.

      A good concise written account of DNA technology is:

      James Watson, Michael Gilman, Jan Witkowski & Mark Zoller. Recombinant DNA, 2nd edition. NY: WH Freeman, 1992.