chapter 19

• Molecular genetic analysis
• Chapters covered in exam

  • 8
  • 16-19
  • 20
  • 22-23
• Learning objectives


  • List the key innovations in molecular genetics and what they can be used for
  • Design a forward genetic experiment to find the gene that causes a phenotype
  • Design a reverse genetic experiment to identify the phenotype(s) affected by a gene of interest

  
• Key innovations in molecular genetics

  • Recombinant DNA
  • Amplifying & visualizing DNA
  • Cutting DNA
  • Sequencing DNA

  
• Applications of molecular genetic techniques

  • Identifying a causative gene (forward genetics)
  • Finding genes involved in producing a phenotype (reverse genetics)
  • Genetic engineering - transgenics

  
• Polymerase chain reaction (PCR)


  Used to amplify a specific sequence of DNA

  
• Polymerase chain reaction setup

  1. Denature

  2. Annealing

  3. DNA synthesis

  
• PCR creates millions of copies of a specific segment of DNA in ~hour
• Gel electrophoresis


  Separates DNA by size and charge by running DNA through a gel matrix

  
• Smaller DNA fragments pass more quickly through the gel matrix and migrate further towards the positive electrical node

  
• DNA is stained with a fluorescent dye that binds specifically to DNA and fluoresces in UV light

  
• Reasons for cutting DNA

  • To make recombinant DNA
  • To distinguish DNA sequences (genotype)
  • To induce mutations

  
• Recombinant DNA


  DNA created by combining genetic material from multiple sources

  
• Restriction enzymes are a fast, reliable, and specific way to cut DNA
• Restriction enzyme recognition sequences are short and therefore not unique in the genome
• Targeted mutagenesis


  Inducing a mutation in a specific gene to study the function of that gene
• CRISPR-Cas systems


  Prokaryotic immune systems that cut foreign (bacteriophage) DNA
• Stages of CRISPR-Cas defense

  1. Adaptation

  2. Expression

  3. Interference

  
• PAM sequence is required for CRISPR-Cas systems to cut foreign DNA

  
• CRISPR-Cas9 system


  Evolutionary diverse systems, Type II requires a single Cas protein (Cas9) for expression and interference, so was modified for use in genome engineering

  
• Designing a CRISPR experiment

  1. Choose crRNA sequence

  2. Identify PAM sequence

  
• Targeted mutagenesis using CRISPR-Cas

  1. Deliver CRISPR gRNA and Cas9 to cells

  2. Effector complex binds to complementary sequence in genomic DNA

  3. Cas9 cuts the target sequence creating a double strand break

  4. DNA repair via nonhomologous end joining or homology directed repair

  
• Functions of CRISPR-Cas Systems

  • Cuts double-stranded DNA at specific sequences
  • Cuts single-stranded DNA
  • Cuts single-stranded RNA
  • Cuts double-stranded DNA with high accuracy, very little off-target cleavage
  • Create single base pair substitutions
  • Recognizes more common PAM sequences
  • Activates transcription
  • Inhibits transcription
  • Induces multiple mutations within a short section of DNA
  • Detects pathogen DNA or RNA
  • Creates a retrievable record in DNA

  
• Sanger sequencing


  Used to sequence a specific DNA sequence

  
• Sanger sequencing


  1. Add template DNA & only 1 primer

  2. Add dNTPs & DNA polymerase

  3. Add a different ddNTP to each of 4 reactions

  
• CAMERA


  Detects pathogen DNA or RNA

  
• CRISPR-Cas9 systems


  Creates a retrievable record in DNA

  
• Key innovations in molecular genetics

  • Recombinant DNA
  • Amplifying & visualizing DNA
  • Cutting DNA
  • Sequencing DNA

  
• Applications of molecular genetic techniques

  • Identifying a causative gene (forward genetics)
  • Studying gene function by observing phenotypic effects of genetic manipulations (reverse genetics)
  • Genetic engineering - transgenics
• Uses of Sanger sequencing

  • Genotype individuals for a known sequence variation
  • Compare sequences across individuals or species to infer evolutionary history
  • Identify a disease-causing mutation in a gene

  
• Sanger sequencing


  1. Template DNA & only 1 primer

  2. Add dNTPs & DNA polymerase

  3. Add a different ddNTP to each of 4 reactions

  4. ddNTPs are sometimes incorporated instead of the corresponding dNTP causing synthesis of that molecule to stop

  
• Dideoxynucleotides


  Missing the 3'-OH group required to form phosphodiester bond during DNA synthesis
• Automated Sanger sequencing


  1. ddNTPs are labeled with fluorescent dyes

  2. All ddNTPs are added to a single reaction

  3. The end products are run through a capillary electrophoresis in a sequencing machine

  4. A laser excites the dyes and a detector records the fluorescence as the DNA molecules pass by

  
• Next-generation sequencing (NGS)


  Product of NGS are short reads (50-100bp) spread across the genome
• Next-generation sequencing


  1. Fragment the entire genome

  2. Add adapters

  3. Wash fragments onto flow cell

  4. Adapters bind to flow cell oligos

  5. Amplify to make clusters

  6. Sequencing by synthesis using fluorescently labeled nucleotides

  7. Fluorescence is detected for each cluster

  
• Sanger sequencing is locus specific and relies on a single primer that you design to the locus of interest

  
• The first draft of the human genome was sequenced using Sanger sequencing

  
• Sanger sequencing took 13 years and $3 billion to sequence the first human genome

  
• Forward genetics


  Identify the gene that causes a specific phenotype
• Forward genetics techniques


  • Linkage analysis to known mutants or genetic markers
  • Sequencing
  • Genome-wide association studies (GWAS)
  • Quantitative trait locus mapping (QTL)