Making DNA fragments

• Creating DNA fragments is used is many different processes such as; sequencing , genetically modifying organisms and genetic fingerprinting.
• There are multiple methods that can be used to isolate a target gene;

  • using reverse transcriptase enzymes to make DNA from mRNA
  • Use of restriction enzymes to cut out a section of DNA from the genome
  • the use of a gene machine to build a section of DNA using nucleotides.
• Using Reverse transcriptase to make cDNA;

  1. Reverse transcriptase is an enzyme that can convert RNA back into DNA it has been isolated from a retrovirus such as HIV.
  2. The reverse transcriptase enzyme is added and uses free DNA nucleotides to make DNA from the mRNA template this is called cDNA.
  3. addition of DNA polymerase to make it double stranded
• Restriction endonucleases are a group of enzymes that can recognize and cut hydrogen bonds at specific sequences of DNA.
• Restriction enzymes cut at a specific recognition sequence in the DNA which is complementary to their active site. if there are recognition sites at each end of the gene you want you can cut out your gene from the rest of DNA.
• Restriction enzymes form sticky ends and allow different fragments to be joined together or annealed. the restriction sequence is palindromic , they are complimentary to each other.
• Using a gene machine to build fragments:

  1. the scientist first examine the protein of interest to identify the amino acid sequence and form that work out what the mRNA and DNA sequence would be.
  2. the sequence is designed or fed into the computer , the sequence is checked for biosafety.
  3. the computer designs a series of small ,overlapping single strands of nucleotides which can be assembled into the desired gene.
  4. in an automated process each of nucleotides are joined together to form a gene.
  5. Using sticky ends the genes can then be inserted into a bacterial plasmid which acts as a vector.