Making DNA Fragments

• What does recombinant DNA technology allow?

  Allows us to combine genetic material from different sources. The first step in recombinant DNA technology is often making a DNA fragment
• What is DNA fragment?

  A bit of DNA containing a gene
• What does Recombinant DNA technology involve?

  Transferring a fragment of DNA from one organism to another.
• Why can transferred DNA be used to produce of protein in the cells of the recipient organism?

  Because the genetic code is universal (the same DNA base triplets code for the same amino acids in all living things), and because transcription and translation mechanisms are pretty similar too
• What are organisms that contain transferred DNA known as?

  transgenic organisms
• In order to transfer a gene from one organism to another, first need to get a DNA fragment containing the gene you're interested in (target gene)
• Method using reverse transcriptase


  1. Isolate mRNA from cells

  2. Mix mRNA with free DNA nucleotides and reverse transcriptase

  3. Reverse transcriptase uses mRNA as template to synthesise new strands of cDNA
• Most cells

  • Only contain 2 copies of each gene - makes it difficult to obtain a DNA fragment containing the target gene
• Cells that produce the protein coded for by the target gene

  • Will contain many mRNA molecules that are complementary to the gene - mRNA is often easier to obtain
• mRNA molecules

  Can be used as templates to make lots of DNA
• Reverse transcriptase

  Makes DNA from an RNA template - DNA produced is complementary DNA (cDNA)
• Palindromic sequences


  Sequences of nucleotides that read the same forwards and backwards
• Antiparallel base pairs

  Base pairs that read in opposite directions
• Restriction endonucleases

  Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
• Restriction endonucleases

  • EcoRI cuts at GAATTC
  • HindIII cuts at AAGCTT
• Using restriction endonucleases to separate a DNA fragment

  1. DNA sample is incubated with the specific restriction endonuclease

  2. Enzyme cuts the DNA fragment out via a hydrolysis reaction

  3. Sometimes the cut leaves sticky ends (small tails of unpaired bases at each end of the fragment)

  4. Sticky ends can be used to anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
• Gene Machine?

  More recently, technology has been developed so that fragments of DNA can be synthesised from scratch, without the need for pre-existing DNA templates. Instead, a database contains the necessary information to produce the DNA fragment. This means that the DNA sequence does not have to exist naturally - any sequence can be made
• How a gene machine works

  1. Sequence required is designed (if one doesn't already exist)

  2. First nucleotide in the sequence is fixed to some sort of support e.g. a bead

  3. Nucleotides are added step by step in the correct order, in a cycle of processes that includes protecting groups

  4. Protecting groups make sure that the nucleotides are joined in the right points, to prevent unwanted branching

  5. Short sections of DNA called oligonucleotides, roughly 20 nucleotides long are produced

  6. Oligonucleotides are broken off from the support and all the protecting groups are removed

  7. Oligonucleotides can then be joined together to make longer DNA fragments