HG week 11 lab

Electrophoresis Controls

  • Lambda DNA digested with HindIII is a commonly used DNA ladder and can be used as a positive control.
  • Undigested Lambda DNA can be used as a negative control.
  • Wells are prepared for:
    • Positive control
    • Negative control
    • Your samples

Electrophoresis Results

  • Shows variation in success across different wells.
  • Wells are labeled 1-11, including:
    • Positive control
    • Negative control
    • Your samples

Polymerase Chain Reaction (PCR) Review

  • PCR is a targeted form of DNA replication.
  • Key ingredients include:
    • DNA
    • Buffer
    • dNTPs
    • Primers
    • Taq polymerase
  • Occurs across three distinct temperature steps:
    • Denaturation
    • Annealing
    • Extension
  • Cycles are repeated approximately 30 times to allow for an exponential increase in target DNA.

Our PCR Target – a VNTR

  • The human genome is filled with repetitive DNA.
  • VNTR = Variable Number of Tandem Repeats
    • VNTRs are generally sequences of between 10-100bp that are repeated alongside one another down the chromosome
  • Repeats are not always the same and are alongside/adjacent to one another.

Our PCR Target – a VNTR

  • Mistakes in copying VNTR DNA are not efficiently fixed.
  • The number of copies of the VNTR is highly variable, both between people and chromosomes.
  • VNTRs are still inherited 50/50 from parents.
  • Because of their variability, they make excellent targets for individual identification (e.g., Forensics).

VNTR Amplification

  • Primers are designed to sit on either side of the VNTR.
  • The entire repetitive DNA sequence will be amplified.
  • The size of the PCR product generated will be dependent upon how many repeats are between the two primers.
  • VNTRs with more repeats will produce bigger fragments than those with fewer repeats.
  • Example:
    • 5 VNTR repeats will produce a smaller PCR fragment.
    • 10 VNTR repeats will produce a larger PCR fragment.

VNTR Electrophoresis

  • The ApoB VNTR has multiple alleles, with more than 10,000 unique combinations possible.
  • The repeat sequence is 15 bp: TTATAAAATATTTAA.
  • A wide array of different banding patterns will be observed.

Experimental Protocols

  1. Label a 1.5 mL tube and all 250 mL of NET buffer.
  2. Rinse mouth with saline to remove any food particles and other contaminants.
  3. Gently scrape the inside of the cheek with the blunt end of a toothpick to collect cells.
  4. Twirl the toothpick in the NET buffer to remove cells and then discard.
  5. Centrifuge for 1 minute @ 13,000 rpm.
  6. Remove and discard 230 mL of supernatant.
  7. Add 250 mL of fresh NET buffer and resuspend your cells with a pipette.
  8. Centrifuge for 1 minute @ 13,000 rpm.
  9. Remove and discard 240 mL of supernatant.
  10. Add 250 mL of Tween-20 and resuspend your cells with a pipette.
  11. Incubate on ice for 20 minutes.
  12. During this incubation, DNA necklaces can be made.

Experimental protocols: Reagent Quantities

  • Cheek cell DNA protocol vs. Control DNA protocol
  • Sterile water:
    • Cheek cell DNA protocol: 4 mL4 \, \text{mL}
    • Control DNA protocol: 8 mL8 \, \text{mL}
  • 2x Apo-B reaction mix:
    • Cheek cell DNA protocol: 10 mL10 \, \text{mL}
    • Control DNA protocol: 10 mL10 \, \text{mL}
  • Buccal cell extract (your DNA):
    • Cheek cell DNA protocol: 5 mL5 \, \text{mL}
    • Control DNA protocol: -
  • VNTR control DNA:
    • Cheek cell DNA protocol: -
    • Control DNA protocol: 1 mL1 \, \text{mL}
  • Taq Polymerase:
    • Cheek cell DNA protocol: 1 mL1 \, \text{mL}
    • Control DNA protocol: 1 mL1 \, \text{mL}
  • Total Volume:
    • Cheek cell DNA protocol: 20 mL20 \, \text{mL}
    • Control DNA protocol: 20 mL20 \, \text{mL}