L10: PCR and Sequencing

• What is the direction of DNA synthesis?

  DNA synthesis occurs in the 5’ to 3’ direction.
• What does it mean that DNA strands are anti-parallel?

  It means that one strand runs 5’ to 3’ while the other runs 3’ to 5’.
• What is the significance of Watson-Crick base pairing in DNA replication?

  It ensures accurate pairing of nucleotides during DNA replication.
• Who developed the method for DNA sequencing based on partial chemical degradation?

  Walter Gilbert developed this method.
• What is the main principle behind Sanger sequencing?

  Sanger sequencing relies on the incorporation of dideoxynucleotides into newly replicated DNA.
• Why are dideoxynucleotides considered chain terminators in DNA sequencing?

  They lack a 3’-OH group, preventing further nucleotides from being added.
• What are the key components of Sanger's dideoxy sequencing method?

  • Single-stranded template DNA
  • A primer complementary to the template
  • DNA polymerase
  • A pool of normal deoxynucleotides (dATP, dTTP, dGTP, dCTP)
  • A small proportion of radioactively-labelled ddATP
• What is the purpose of electrophoresis in Sanger sequencing?

  It separates the nested fragments of DNA based on size.
• Who invented the Polymerase Chain Reaction (PCR)?

  Kary Mullis invented PCR in 1983.
• What is the primary function of PCR in molecular biology?

  PCR amplifies a specific DNA sequence.
• What were some key milestones in the history of DNA research leading up to PCR?

  • 1953: Watson and Crick propose the DNA model.
  • 1957: Kornberg discovers the first DNA polymerase.
  • 1960s: Khorana deciphers the genetic code.
  • 1969: Brock isolates Thermus aquaticus.
  • 1976: Taq polymerase is isolated.
• Why is Taq polymerase particularly useful for PCR?

  It is thermally stable, allowing it to function at high temperatures.
• What are the three main steps in the PCR process?

  Denaturation, annealing of primers, and extension of primers.
• How does the number of DNA copies change with each cycle of PCR?

  Each cycle doubles the number of copies of the target DNA.
• What is the technique used to visualize PCR reactions?

  • Agarose gel electrophoresis
  • DNA migrates towards the positive terminal due to its negative charge
  • The gel contains a dye that fluoresces under UV light
• What is the visibility of DNA after 30 cycles of PCR?

  After 30 cycles, the DNA is clearly visible.
• What are the specificity and sensitivity of PCR?

  • Specificity: Provided by primers that are complementary to the target sequence.
  • Sensitivity: One target molecule can be amplified to over $10^9$ molecules in hours.
• What are some applications of PCR?

  PCR is used to amplify tiny amounts of DNA for various molecular biology techniques.
• What is the composition of the gel used in agarose gel electrophoresis?

  Agarose
• Why does DNA migrate towards the positive terminal during electrophoresis?

  Because DNA is negatively charged
• How does the size of DNA fragments affect their migration speed in agarose gel electrophoresis?

  Large fragments migrate slowly while small fragments migrate quickly
• What technique is used to visualize PCR reactions?

  Agarose gel electrophoresis
• What happens to the visibility of DNA after 30 cycles of PCR?

  It becomes clearly visible
• What are the two key features of PCR?

  • Specificity: Provided by complementary primers targeting opposite strands
  • Sensitivity: Amplifies one target molecule to over $10^9$ molecules in hours
• What are some applications of PCR in molecular biology?

  Amplifying tiny amounts of DNA for analysis, including DNA sequencing and cloning
• How can PCR be used for prenatal genetic screening?

  By analyzing small samples like chorionic villus samples or a drop of blood
• How can mutations be introduced into amplified DNA during PCR?

  By engineering mismatches in a primer close to its 5' end
• What was the conclusion of the study by Green et al. regarding Neanderthal DNA?

  Europeans and Asians have ~4-5% of their genes derived from Neanderthals
• What does the absence of mtDNA gene flow between Neanderthals and modern humans suggest?

  It suggests male Neanderthal and female human couplings
• What significant findings were made regarding Denisovan fossils?

  • DNA sequenced from teeth and bones
  • Denisovans found in Tibet and Laos
  • Contributes to understanding of human evolution
• What does the term 'forensic' refer to in forensic science?

  It refers to legal applications
• Who first used VNTRs for DNA profiling?

  Sir Alec Jeffreys
• What is a Variable Number Tandem Repeat (VNTR)?

  It is a run of short repeated nucleotide sequences
• How do STRs contribute to personal identification?

  Each STR acts as an inherited allele, showing variations between individuals
• What is the purpose of multiplex PCR?

  To perform multiple reactions in one tube using specific primers for different STRs
• How does using multiple STRs increase discrimination in forensic analysis?

  It allows for more unique combinations of alleles to identify individuals
• What is the outcome for the Lady in the forensic scenario presented?

  She is found innocent