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Day 5 Afternoon Lecture Notes

Steve Williams, Smith College

June 10, 2004

Different RevT genes from the family have sequences that are about 90% the same. When genes are copied from one part of the genome to another, they start to diverge evolutionarily. Most copies of RevT are non-functional "pseudogenes."

RevT codes for reverse transcriptase, which copies RevT to other places in the genome. RevT's tendency to copy itself explains why LINE1 has so many occurrences in the genome. Retroviruses may well be RevT genes that escaped from the host genome. "RevT is something like an endogenous retrovirus." If LINE1 by chance inserts into genes, it may cause a mutation in protein production.

Primer Design Considerations

  1. Desirable length is 18-30 nt. Shorter primers are not specific enough; longer primers don't work any better.
  2. Base composition should be near random as a primer too rich in one base is likely not to hybridize specifically enough.
  3. Pick primers that don't form hairpins. These can be excluded using a computer program. A GC-rich hairpin is more stable (and worse) than an AT-rich one. For example

    GCTACGGGGCCCCTGTG

    would likely fold in the center forming four G-C bonds and fail to hybridize with template DNA.

  4. Avoid primer-primer hybridization. Hairpins are an example of intraprimer hybridization. Another example is when reverse and forward primers bind to each other.
  5. Melting temperatures of the primers should be close. Primers should be hybridized under fairly stringent conditions at (Tm - 10°C) <= T <= (Tm - 5°C). Too low a temperature may cause mishybridization.
  6. Some mismatch of the primer and template can be tolerated but not at the 3' end. The 3' end is where synthesis and elongation start, so if it is dangling, these processes will fail.

Forensic investigations use standard primers that hybridize to highly conserved DNA regions but which, taken as a pair, span highly variable regions. Some primer and software vendors: Molecular Biology Insights (including the Oligo program), DNAsis, LaserGene, IDT (Smith's provider) and the Primer3 program from MIT.

How to Confirm the Identity of PCR Products

  1. A band on a gel at the right mass is an indication of the right product. For a routine process using a gel to check the mass is adequate. For a new PCR process with new primers, more verification is called for.
  2. Use an internal hybridization probe.

    3. Design a 3rd nucleotide that hybridizes to the known sequence of the expected PCR product. Often this test is performed using a Southern blot: running a DNA gel, then transferring DNA to a membrane where hybridization with the probe is checked.

    The buffer goes up the wick, through the gel, through the membrane and into the paper. DNA follows the buffer through the gel and onto the membrane, which it can't permeate. In so doing it moves purely vertically and stays registered with its position in the gel. The DNA transfer is always performed with the gel upside-down as the top surface is too wavy to make good contact to the membrane.

    The DNA on the membrane can be UV-crosslinked on for attachment and then hybridized with a probe. The probe can be labeled with fluorescein or biotin. Any DNA in marker lanes will also be transferred to the membrane and will serve as a negative control for the hybridization. Oligos can be ordered with attached probes for added convenience. Any ethidium bromide left in the gel doesn't interfere with the hybridization. Hybridization of a probe to a Southern membrane should used high stringency since the method is intended to be a strict confirmation of earlier results.

  3. Perform a second PCR reaction with different primers from the first one. A second PCR reaction is a better test than Southern blot as it requires the matching of two new oligos. This method is known as "nested" PCR.
  4. Perform a restriction enzyme digest. If the PCR product is the desired one, the products on the gel should appear at predictable masses. The restriction enzyme method used to be known as the "DNA fingerprint" although it has nothing to do with the forensic method now known by that name.
  5. Sequence the DNA of the PCR product. Sequencing is now the method of choice now that it's easy and cheap.
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