dna technolog

• name 3 ways to make dna fragments

  • restriction enzymes
  • gene machine
  • reverse transcriptase
• how to make DNA fragment using restriction enzymes

  • cuts double stranded dna at specific recognition sites which are palindromic
  • sample is incubated with restriction endonuclease which cuts DNA by hydrolysing the phosphodiester backbone
  • can cut straight down the middle creating blunt ends or can cut zig zagged to form sticky ends
• how to make DNA fragment using reverse transcriptase

  • mRNA is isolated from cell and mixed w/ free DNA nucleotides and reverse transcriptase
  • reverse transcriptase uses the mRNA as a template to synthesise a new strand of dna
• why do you use MRNA in fragmentation than DNA using reverse transcriptase

  mrna is easier to extract as there is more copies

  mrna is complementary to the DNA
• how to obtain DNA fragments using gene machine

  • nucleotide is fixed onto a solid support
  • nucleotides individually added in the correct order
  • protection groups are added to prevent unwanted branching of the chain
  • oligonucleotides are synthesised , they are broken off of the support and protecting groups are removed.
  • oligonucleotides are added together to make longer dna fragments
•  Describe the roles of two named types of enzymes used to insert DNA fragments into plasmids.

  •   Restriction endonuclease to cut plasmid/vector
  •       Ligase joins gene/DNA to plasmid/vector
• DNA probe

  A short, single-stranded length of DNA that has a marker/label attached that makes it easily identifiable
• Types of DNA probes

  • Radioactively labelled probes
  • Fluorescently labelled probes
• Radioactively labelled probes

  Made up of nucleotides with the isotope phosphorous-32 attached, identified using X-ray film exposed by radioactivity
• Fluorescently labelled probes

  Emit light (fluoresce) under certain conditions, when the probe has bound to the target DNA sequence
• Using DNA probes to identify particular alleles of genes

  1. Make DNA probe with base sequences complementary to part of the base sequence of the allele

  2. Separate double-stranded DNA being tested into two strands

  3. Mix strands with probe, which binds to complementary base sequence

  4. Identify site where probe binds by radioactivity or fluorescence
• DNA hybridisation

  The process where the DNA probe binds to the complementary base sequence on one of the DNA strands