Manipulating genomes

• What is the first step in DNA sequencing?

  Mapping the existing information about the genome
• How is DNA fragmented in the sequencing process?

  Using restriction enzymes

  
• What is formed after DNA is fragmented and inserted into bacterial artificial chromosomes?

  A genomic DNA library
• What happens to the fragments obtained from bacterial cultures during DNA sequencing?

  They are broken down into smaller fragments
• What technique is used to sequence the smaller DNA fragments?

  Chain-termination method developed by Sanger
• What is the role of DNA polymerase in the chain-termination method?

  It incorporates chain-terminating nucleotides into a growing chain
• How are the four separate sequencing reactions set up in DNA sequencing?

  Each contains all four standard nucleotides, DNA polymerase, primers, and modified nucleotides
• What happens when a modified nucleotide is incorporated into a growing DNA chain?

  Replication is terminated
• What technique is used to separate DNA fragments by size?

  High resolution electrophoresis

  
• How are DNA fragments visualized after electrophoresis?

  Under UV light

  
• What has the rapid advancement of sequencing techniques allowed for?

  High-throughput sequencing and whole genome sequencing
• Why is comparing genomes between species significant?

  It allows evolutionary relationships to be determined
• How does gene sequencing contribute to personalized medicine?

  It identifies differences that can be tailored to specific genomes
• What has gene sequencing allowed for in terms of polypeptides?

  Prediction of the sequences of amino acids
• What is DNA profiling used for?

  To identify individuals by characteristics of their DNA
• What is the main technique used in DNA profiling?

  Polymerase chain reaction (PCR)
• What is the purpose of PCR in DNA profiling?

  To amplify the DNA by making millions of identical copies
• What are the steps involved in setting up a PCR reaction mixture?

  Mixing DNA sample, primers, free nucleotides, and DNA polymerase
• What temperature is the PCR mixture heated to in order to break hydrogen bonds?

  95 degrees Celsius
• At what temperature do primers bind to the DNA strands during PCR?

  Between 50-65 degrees Celsius
• What is the optimum temperature for DNA polymerase to work during PCR?

  About 70 degrees Celsius
• How many times is the PCR cycle typically repeated?

  Around 30 times
• What is gel electrophoresis used for in DNA profiling?

  To separate DNA fragments and proteins according to their size

  
• Why do DNA fragments move through the gel during electrophoresis?

  Because they have a negative charge

  
• What are the main steps in the DNA sequencing process?

  1. Mapping the genome
  2. Fragmenting DNA with restriction enzymes
  3. Inserting fragments into bacterial artificial chromosomes
  4. Creating a genomic DNA library
  5. Sequencing using chain-termination method
  6. Visualizing fragments under UV light

  
• What are the applications of gene sequencing?

  • Genome-wide comparisons between individuals and species
  • Determining evolutionary relationships
  • Medical research benefits
  • Development of personalized medicine
  • Prediction of amino acid sequences in polypeptides
  • Advancements in synthetic biology
• What are the steps involved in the PCR process?

  1. Set up reaction mixture with DNA sample, primers, nucleotides, and DNA polymerase
  2. Heat to 95°C to separate strands
  3. Cool to 50-65°C for primers to bind
  4. Increase to 70°C for DNA polymerase activity
  5. Repeat cycle around 30 times
• What are the main techniques used in DNA profiling?

  • Polymerase chain reaction (PCR)
  • Gel electrophoresis

  
• What is the significance of DNA profiling in forensic science?

  • Identifies individuals by DNA characteristics
  • Determines genetic relationships between organisms