Lab PCR 1

• Lab coats are required. No contact lenses. No food or drink. Leave your coats and backpack in your locker.
• Week 1: PCR


  Amplify "Short Tandem Repeats" (STR's) region of DNA from two individuals suspected of committing a crime, as well as the DNA found at the crime scene
• Week 2: Electrophoresis


  Separate the STR samples amplified the week before and identify which of the suspects' DNA was found at the crime scene
• Pre-Lab work: Read through the lab instructions BEFORE the lab.
• Pre-Lab work: Consult the following virtual demonstration of PCR. Click on Amplification to start the demo. Click on blue box to make the demo move forward.
• Pre-Lab work: Consult the following online tutorial on DNA electrophoresis. Click on arrow to make the demo move forward.
• DNA replication in the live cell


  Compared to the PCR method
• DNA fingerprinting using Short Tandem Repeats (STR's)


  Compared to DNA fingerprinting using RFLP's
• Restriction endonucleases


  Role in cutting DNA for RFLP preparations

  
• Thermal cycler


  How it operates and identify the importance of Taq DNA polymerase enzyme in PCR amplification

  
• Gel electrophoresis


  Principle of separation of DNA molecules by charge and size
• Conditions in gel electrophoresis


  • Which can affect the resolution of the band separation
• Preparing agarose gels for electrophoresis


  Prepare chamber buffer and load the gel
• Interpret experimental results and identify the criminal responsible for a fictitious crime.
• DNA fingerprinting


  Technique that can be used to identify and distinguish between individuals and determine ancestral relationships
• Restriction Fragment Length Polymorphisms (RFLP)


  Early DNA fingerprinting technique that uses restriction endonucleases to create DNA fragments of varying lengths
• RFLP method is accurate but have the disadvantage of requiring a lot of starting genetic material
• Tandem repeats


  Regions of the genome composed of tandemly repeated motifs of varying length, classified into short tandem repeats (STRs) and variable-number tandem repeats (VNTRs)

  
• Short tandem repeats (STRs)


  Suitable for human identification due to the variable number of repeats between people
• STRs have become popular DNA markers because they are easily amplified by polymerase chain reaction (PCR)

  
• The world standard in forensic human identification (CODIS) relies on using 13 independent loci (chromosome positions) where STRs are known to be present
• The probability of a random match when all 13 CODIS loci are analyzed is 1 in 3 trillion, except for identical twins
• Performing STR analysis


  Extraction of nuclear DNA, amplification of certain regions using PCR, separation by gel electrophoresis to determine number of repeats

  
• Polymerase chain reaction (PCR)


  Amplifies DNA of interest to a measurable quantity, using Taq polymerase, primers, and free nucleotides

  
• PCR steps


  Denaturation, annealing, extension - each cycle doubles the amount of targeted DNA
• DNA denaturation in PCR

  Heating the sample to 94°C breaks the hydrogen bonds between the base pairs
• DNA primers in PCR


  Short synthetic pieces of DNA that target a specific part of the genome, forward and reverse primers used to replicate both DNA strands

  
• PCR cycle


  1. Denaturation: 94°C for 30 seconds

  2. Annealing: 45°C for 65 seconds

  3. Extension: 72°C for 30 seconds
• Denaturation


  Breaking the hydrogen bonds between the base pairs to separate the two complementary strands of DNA

  
• Annealing


  Allowing the primers to bind to the target DNA sequence

  
• Extension


  DNA polymerase synthesizing the new complementary DNA strand

  
• Taq polymerase


  The key enzyme used during PCR

  
• Taq polymerase was first discovered in the bacterium Thermus aquaticus, which was found living within a thermal spring
• Taq polymerase


  It can withstand high temperatures, which is perfect for PCR
• Ingredients required in PCR tubes


  • Specific DNA sample
  • Primers (forward and reverse)
  • Master mix (Taq polymerase, MgCl2, dNTPs, reaction buffer)