Manipulating Genomes

• Who developed the controlled termination method for DNA sequencing?

  Frederick Sanger
• What is the first step in Sanger Sequencing?

  Convert DNA into single stranded form
• How are complementary primer sequences obtained in Sanger Sequencing?

  From restriction digest or gene machine
• What is the role of the dideoxy analogue in Sanger Sequencing?

  It blocks further chain growth

  
• What happens when DNA polymerase copies the DNA half strand in Sanger Sequencing?

  A series of fragments are produced
• How many times is the Sanger Sequencing process repeated?

  Three more times
• What is the purpose of electrophoresis in Sanger Sequencing?

  To separate DNA fragments by size

  
• What is increasingly replacing radioactive labeling in DNA sequencing?

  Fluorescent tagging
• What is pyrosequencing known for?

  Its speed and accuracy
• What is the first step in pyrosequencing?

  Fragmenting and amplifying the DNA sample

  
• What role does ATP sulfurylase play in pyrosequencing?

  It converts PPi to ATP
• How is light produced in pyrosequencing?

  By luciferase using ATP as substrate

  
• What does the intensity of light signal indicate in pyrosequencing?

  Number of nucleotides incorporated

  
• What was the primary goal of the Human Genome Project?

  To determine the sequence of human DNA
• How many base pairs make up human DNA?

  Three billion
• What does comparing DNA sequences tell us about organisms?

  Phylogenetic relationships
• Why is coding DNA used for classification?

  It is similar within species, different between
• What can be deduced from the amino acid sequence of proteins?

  The corresponding DNA sequence
• What is synthetic biology?

  An interdisciplinary field combining biology and engineering
• What is CRISPR-Cas9 used for?

  Gene editing
• What does gel electrophoresis separate?

  DNA or protein fragments by size

  
• What is the purpose of DNA markers in gel electrophoresis?

  To determine sizes of unknown DNA
• What is restriction mapping used for?

  Aiding sequencing of large DNA pieces
• How does a DNA probe work?

  It binds to complementary DNA sequences
• What is the purpose of Southern blotting?

  To transfer DNA fragments to a membrane

  
• What are minisatellites in DNA fingerprinting?

  Repetitive non-coding base sequences
• What are the steps involved in Sanger Sequencing?

  1. Convert DNA to single stranded form
  2. Add complementary primers
  3. Add nucleotide triphosphates and dideoxy analogue
  4. Polymerase copies DNA, producing fragments
  5. Repeat with different dideoxy analogues
  6. Electrophorese fragments to analyze results

  
• What are the steps involved in pyrosequencing?

  1. Fragment and amplify DNA sample
  2. Prepare enzyme mixture
  3. Add activated nucleotide to DNA template
  4. Incorporate nucleotide, releasing PPi
  5. Convert PPi to ATP and produce light
  6. Record light signals to determine sequence

  
• What are the uses of DNA sequencing?

  • Human Genome Project
  • Classification of organisms
  • Understanding phylogenetic relationships
  • DNA fingerprinting
• What are the fundamental techniques in synthetic biology?

  • DNA Synthesis
  • CRISPR-Cas9 gene editing
  • Gene Circuits design
• What are the applications of DNA probes?

  • Screening for specific genes
  • Painting chromosomes
  • Detecting gene expression
  • Identifying mutations
  • DNA microarrays
• What is the purpose of DNA probes in chromosome painting?

  To identify different chromosomes
• How can DNA probes be used to detect gene expression?

  They target mRNA molecules
• What is the role of DNA probes in detecting mutations?

  They identify mutations altering restriction sites
• What are DNA microarrays used for?

  • Thousands of probes on a chip
  • Hybridize with target DNA or RNA
  • Probe for multiple genes or mutations simultaneously

  
• What are minisatellites and microsatellites in DNA fingerprinting?

  Repetitive non-coding base sequences
• Why do minisatellites and microsatellites vary between individuals?

  They accumulate mutations harmlessly over time
• What is the probability of a DNA fingerprint match between two random individuals?

  1 in 28 million
• How can DNA fingerprinting be used in paternity disputes?

  It analyzes slight differences in DNA
• What is the first step in DNA fingerprinting analysis?

  PCR to amplify the DNA sample