Recombinant DNA

• Recombinant DNA technology


  technology that combines genes from different organisms into a single DNA molecule
• What is this recombinant DNA technology used for?


  By scientists to manipulate and alter genes to improve industrial processes and medical treatment
• What is the first step of recombinant DNA technology?


  To produce or isolate fragments of DNA to be recombined with another piece of DNA
• Three methods of isolating fragments of DNA

  1. Reverse transcription

  2. Restriction endonucleases

  3. Gene machine
• Process of reverse transcription


  1. Reverse transcriptase can make DNA copies from mRNA

  2. Naturally occurs in viruses such as HIV

  3. A cell that naturally produces the protein of interest is selected

  4. These cells should have large amounts of mRNA for the protein
• last steps of reverse transcription

  5. The reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence

  6. Single stranded DNA is made (cDNA)

  7. To make this DNA fragment double stranded you add more nucleotides and DNA polymerase

  
  

  (Last step is PCR)
• Advantage of reverse transcription


  cDNA is intron free because it is based on the mRNA template
• Restriction endonucleases


  an enzyme that cuts DNA at specific sites, producing small fragments used in genetic engineering.
• What do restriction endonucleases have?


  An active site complementary in shape to a range of different DNA base sequences, described as recognition sequences, and therefore each enzyme cuts the DNA at a specific location
• Blunt end


  Some enzymes cut at the same location in the double strand, and create a blunt end.
• Sticky ends


  Enzymes cut to create staggered ends and exposed DNA bases. These are palindromic and have the ability to join DNA with complementary base pairs
• What can these sticky ends be used for?


  The exposed bases can be aligned with the bases for the organism you want to insert it into. Makes it easier to join the DNA
• Gene machine


  DNA fragments can be created in a lab using a computerised machiene
• What do scientists do before the process starts?


  Scientists first examine the protein of interest to identify the amino acid sequence and from that work out what the mRNA and DNA sequence would be
• What can they then do?

  DNA sequence is entered into the computer which checks for biosafety and biosecurity so that the DNA being created is safe and ethical to produce. The computer can create small sections of overlapping single strands of nucleotides that make up the gene called oligonucleotides
• What can happen with the oligonucleotides?


  Can be joined to create the DNA for the entire gene. PCR can be used to amplify the quantity and make the double strand
• Advantages of the gene machiene?


  Very quick, accurate and makes intron free DNA so can be transcribed in prokaryotic cells
• Pros and cons of reverse transcriptase


  -mRNA present in cell is from actively transported genes, so lots of the MRNA available to make cDNA

  -more steps, so more time consuming and difficult
• Pros and cons of restriction endonuclease


  -Sticky ends on DNA fragment make it easier to insert to make recombinant DNA

  -still contains introns
• Pros and cons of gene machines


  -Can design exact DNA fragment you want with sticky ends, labels and preferential codons

  -need to know the sequence of amino acids or bases
• in vivo cloning-what happens before


  Restriction endonuclease is used to cut out the DNA fragment/gene of interest. These leave sticky ends. The DNA fragment must be modified to ensure transcription of these genes can occur
• What must be added?


  A promotor region-added at the start of the DNA fragment. This is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur
• What else must be added?

  A terminator region. Added at the end of the gene. It causes RNA polymerase to detach and stop transcription so that only one gene at a time is copied into mRNA
• What is the next step?

  Insert DNA into a vector
• What is a vector

  Something to carry the isolated DNA fragment into the host cell. Plasmids are the most common vectors. Circular DNA which is small so only has a few genes
• What would you do?


  Plasmid is cut open using the same restriction endonuclease. This creates the same sticky ends. Therefore the DNA fragment sticky ends are complementary to the sticky ends on the plasmid
• What is the final step of the in vivo cloning?


  DNA fragment and cut plasmid are combined and enzyme ligase sticks them together. Ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides
• How to transform a host cell with plasmid?


  -Vector needs to be inserted into the host cell

  -The cell membrane of the host cell must be made more permeable

  -The host cells are mixed with Ca2+ and are heat shocked (sudden increase in temperature)

  -enables vector to enter the host cells cytoplasm
• Why do we have to identify transformed cells?


  Not all host cells will successfully take up the recombinant plasmid?
• What 3 issues can occur?

  1. The recombinant plasmid doesn't get inside the cell

  2. The plasmid re-joins before the DNA fragment entered

  3. The DNA fragment sticks to itself rathe than inserting into the plasmid
• How do you identify if a bacteria successfully took up recombinant plasmid?

  using a marker gene
• What are the three different marker genes used?


  1. Antibiotic resistance genes

  2. Genes coding for fluorescent proteins

  3. Genes coding for Enzymes
• Antibiotic resistance marker gene

  a gene that produces a protein that provides cells expressing this protein with resistance to an antibiotic ????
• Fluorescent markers


  Jellyfish contain a gene which codes to create a green fluorescent protein (GFP). This can be inserted into the bacteria plasmid
• What happens with the fluorescent markers?

  DNA fragment is inserted in the middle of the GFP gene. This disrupts it and prevents GFP production
• What can you then do?

  Grow colonies on agar bacteria, and when exposed to UV light only non-glowing colonies contain the recombinant plasmid
• Lactase-can turn blue substances colourless (Enzyme markers)


  DNA fragment is inserted into enzymes gene to disrupt it. Bacteria are grown on an agar plate with the colourless substance. Colonies which cannot turn the colourless substance blue contain recombinant plasmid as lactase must have been disrupted
• How do you grow multiple copies of the host cell


  Fermenters. This large cloned population can produce the protein coded for by the inserted DNA fragment
• Amplifying of DNA fragments


  Occurs once DNA fragments have been isolated, they need to be cloned to create large quantities either done in vivo or in vitro
• in vitro

  Fragments of DNA are amplified via the polymerase chain reaction by an automated machine