Making DNA fragments

• Recombinant DNA technology


  The transfer of fragments of DNA from one organism or species to another
• Why recombinant DNA technology works

  • Genetic code is universal
  • Transcription and translation mechanisms are universal
  • Transferred DNA can be translated (protein synthesis) within cells of the recipient organism
• Reverse transcriptase


  • Enzyme which can make copies of DNA from mRNA
  • Naturally occurring in viruses - HIV
• How reverse transcriptase can be used to make DNA fragments

  1. A cell which naturally produces the protein of interest is selected

  2. These cells will contain large amount of mRNA to make that particular protein

  3. The reverse transcriptase enzyme joins the DNA nucleotides using complementary base sequences to the mRNA sequence

  4. Single strand of DNA is created - known as cDNA

  5. To make this fragment double stranded enzyme DNA polymerase can be used
• Advantages of cDNA


  • Only contains introns
  • As it's a copy of mRNA after splicing
  • An advantage when genetic engineering involves prokaryotes - which don't have the ability to remove introns
• Restriction endonuclease


  • Enzymes which cut DNA
  • Naturally occur in bacteria as a defence mechanism
• Why it's an advantage for bacteria to have restriction endonuclease


  • Defence mechanism
  • Defend against viral pathogens
• How and why there are different types of restriction endonuclease enzymes

  1. Many restriction endonuclease enzymes which cut only at specific DNA base sequences

  2. They have an active site complementary in shape to specific base sequence - known as recognition sites

  3. Therefore, each enzyme cuts only a a specific location on DNA
• Blunt end


  Cut made by some restriction endonuclease enzymes
• Staggered end


  Cut made by some restriction endonuclease enzymes at palindromic sequences which leaves exposed bases
• Palindromic sequences of DNA


  When a complemtery strand of DNA reads the same in the opposite direction
• Sticky ends


  The exposed bases of DNA when restriction endonuclease enzymes cut at palindromic sequences
• Advantage of sticky ends

  • These exposed bases can be aligned next to the organism you want to insert it into
  • Easier to join DNA once it's added
• Gene machine


  Computerised machines used to make DNA fragments in the lab
• First thing to do before placing your sequence in a gene machine

  1. Scientists must examine the protein of interest to identify the amino acid sequence

  2. From there they can work out the the mRNA and DNA sequence
• How a gene machine can be used to make DNA fragments

  1. DNA sequence identified by scientists is entered into a computer which checks for biosafety and biosecurity - makes sure the DNA fragments being made is safe for humans and ethical to produce

  2. The computer makes small sections of overlapping single strands or nucleotides that make up the gene of interest - known as oligonucleotides
• Oligonucleotides


  Small section of single stranded DNA
• How oligonucleotides are turned into a whole fragment

  Each of the small sections are joined together to create the DNA for the entire gene
• Advantage of reverse transcriptase

  • mRNA is already present in cell
  • Being actively transcribed
  • So lots of mRNA of interest to make cDNA
• Disadvantages of reverse transcriptase


  • More steps
  • More time consuming
  • Technically more difficult
• Advantage of restriction endonuclease


  • Sticky ends make it easier to insert when making recombinant DNA
• Disadvantages of restriction endonuclease

  • Fragments still contain intron
  • Can not be transcribed in prokaryotic cells
• Advantages of a gene machine

  • Very quick once the sequence is originally identified
  • Accurate
  • Can be designed to create sticky ends
  • Can be designed to be intron free so it can be transcribed in prokaryotes
• Disadvantages of gene machine

  • Need to invest time into identifying exact DNA sequences for protein of interest