Topic 4: DNA Manipulation

• Gene of interest: the gene we want the bacteria to express: insulin gene
• plasmid vector: a circular ring of DNA that will be used to transport the gene of interest into the bacteria
• Recombinant plasmid: Plasmid that has had the gene of interest inserted into it
• Endonucleases are enzymes that can break the bonds between nucleotides of nucleic acids (cut DNA).

  Restriction enzymes are a type of endonuclease that are produced by bacteria and can cut DNA at specific sequences.
• Ligases are enzymes that join nucleic acid fragments together by helping to create the bonds between a sugar and a phosphate (backbone)
• Many DNA techniques require a large volume of DNA.

  Amplification of DNA creates many copies of an original DNA sample.

  Polymerases are enzymes that join nucleotides together to create nucleic acids.
• DNA Polymerase requires a primer to begin building a DNA strand.

  A primer is a short complementary strand of nucleic acid
• Creating a recombinant plasmid

  The target gene is removed from the DNA strand using restriction enzymes.

  The plasmid is also cut using the same restriction enzyme.

  This means that the target gene and the plasmid have complementary sticky ends and will be easier to join.

  Once the target gene is positioned in the plasmid, ligases join the two pieces of DNA creating a recombinant plasmid.
• Creating a recombinant plasmid

  A) plasmid

  B) target gene

  C) restriction ezyme

  D) DNA ligase

  E) recombinant

  
• Central Dogma: DNA -> RNA -> Protein -> Products
• Plamid Vectors have 4 important DNA Sequences

  • Restriction Endonuclease Sites which allows the gene of interest to be inserted
  • Antibiotic Resistance Genes -which helps in selecting bacteria which have taken up the plasmid
  • Origin of Replication (ORI)- a site for DNA replication to begin in bacteria
  • Reporter gene - genes with an easily identifiable phenotype so the plasmid that has taken up the gene of interest can be identified.
• Selecting the transformed bacteria

  Not all of the bacteria will take up the recombinant plasmid

  To select only the transformed bacteria plasmid vectors often include an antibiotic resistance gene.

  This allows us to only culture the transformed bacteria that will express our gene of interest.
• Human insulin is a quaternary structure and is made of two polypeptide chains.

  Two separate plasmids must be produced and placed in separate bacteria. One for Chain A and one for Chain B.

  When the polypeptides are extracted they are combined to form functional human insulin.
• Reporter genes are used in bacterial transformation to indicate whether the gene of interest in being expressed or not.

  Reporter genes code for proteins that are easily visualised.

  GFP and lac z are commonly used as reporter genes.

  GFP causes bacteria to glow green.

  lac z causes bacteria to turn blue.
• PCR is a technique that rapidly makes many copies of an original DNA sample.

  There are 4 substances necessary for PCR:

  • DNA sample
  • DNA Polymerase to build new DNA strands
  • Nucleotides
  • Primers a short single strand of nucleic acid that acts as the starting point for DNA polymerase.
• The polymerase used in PCR must maintain its shape at very high temperatures.

  Taq polymerase comes from a a bacteria called Thermus aquaticus that lives in hot springs. It does not denature even a temperature of 95°C.
• Steps of PCR

  1. Denaturing: DNA is heated to approximately 95°C to break the hydrogen bonds and form single stranded DNA
  2. Annealing: The sample is cooled to 55°C to allow primers to bind to complementary sequences of the single stranded DNA.
  3. Extending/Elongation: DNA is heated to 72°C which allows Taq polymerase to work optimally. In binds to the primer, which acts as a starting point, an begins synthesizing a new DNA strand.
• CRISPR is less expensive, takes less time, precise, programmable, they edit live cells, and switch genes on and off.
• Cas9: an endonuclease (cuts DNA). Can be programmed to cut at any desired DNA sequence.

  sgRNA/gRNA: guides the Cas9 and tells it where to cut. Changing the sgRNA is how Cas9 is programmed. Scientists can create sgRNA with any sequence to cut at any known target gene.
• The PAM is a very short segment of nucleotides (2-6) on the DNA that signal Cas-9 to stop and check for a complementary DNA sequence to cut.
• Gel electrophoresis:

  
  

  A technique used to separate DNA fragments according to their size.

  • DNA contains a negative charge
  • gel electrophoresis has an electrical current within the gel.
  • At the negative end of the machine are wells where DNA is placed into
  • DNA travels from the negative end to the positive end with the help of the electrical charge.
  • The result shows how long or short the DNA fragments are compared to each other
• In gel electrophoresis:

  DNA ladder can be used to help identify DNA base pair lengths. It is a sample that has known fragment sizes so when electrophoresis is run in the machine, the lengths can be read. The ladder closest to the wells/negative end is the longest and it goes shorter towards the positive end.
• Making the CRISPR-Cas9 complex

  1. Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA that scientists wish to cut.
  2. A Cas9 enzyme is obtained with an appropriate target PAM sequence.
  3. Cas9 and sgRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex.
• CRISPR-Cas9 for gene editing

  1. The sgRNA-Cas9 mixture is injected into a specific cell, such as a zygote
  2. The Cas9 finds the target PAM sequence and checks whether the sgRNA aligns with the DNA
  3. Cas 9 cuts the selected sequence of DNA.
  4. The DNA has a blunt end cut that the cell will attempt to repair.
  5. When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. Scientists may inject particular nucleotide sequences into the cell with the hop that it will ligate (glue) into the gap.