7.1 using gene sequencing

• genome?


  complete set of genetic information and material in the cells of an organism.
• what is dna sequencing?

  identifying base sequences of nucleotide in dna fragment
• Dna samples are amplified by polymerase chain reactions (PCR)
• what is in the reaction mixture of PCR?

  • target DNA (dna to be amplified)
  • free nucleotides
  • dna polymerase (catalyse formation of phosphodiester bonds)
  • primers
• what are primers?

  • short nucleotide base sequence - complimentary to the dna that is going to be amplified.
  • allows dna polymerase to anneal to template strand and begin replication
• Steps of PCR


  • DNA strand heated to 95 degrees
  • separates DNA strands and hydrogen bonds break
  • cooled to 55 degrees
  • allows primers to anneal to DNA strand
  • heated to 70 degrees
  • to activate DNA polymerase and allow nucleotides to join.
• Steps of Gel electrophoresis

  • Dna sample is amplified by PCR
  • Restriction enzyme breaks DNA into fragments
  • Stained with fluorescent dye
  • Potential difference used
  • Dna shows up as bands
  • No of bands links to similarity
• steps of southern blotting

  • DNA sample is amplified by PCR
  • Restriction enzyme breaks DNA into fragments
  • gel placed in an alkali solution to breaks hydrogen bonds between DNA bases so strands separate. ​
  • thin nylon sheet placed on gel
  • placed in bag with probes
  • probes anneal to DNA with complimentary base
  • looked at through x-ray
• what are probes?


  • short single stranded DNA
  • labelled with radioactive phosphorus
  • locates bands on nylon
  • locates specific alleles and base sequences
• uses of amplified DNA samples

  • DNA sequencing (predicts amino acid sequence/ genetic disease)
  • DNA profiling (forensics)
• steps of DNA sequencing (termination):


  • 4 separate reactions containing...
  • template DNA, DNA polymerase, primers, 4 nucleotides
  • PCR occurs in each reaction tube
  • terminator nucleotide added + replication stops
  • DNA fragments separated by size using gel electropheresis
  • viewed under UV
  • strand moved furthest is first base in complimentary strand
• what is satellite DNA?


  • in non coding regions
  • long strands of DNA containing short tandem repeats (STR)
• Steps of DNA Profiling:


  • DNA sample collected and amplified by PCR
  • satellite DNA cut by restriction enzymes to form fragments
  • fragment length depends on length of short tandem repeats
  • fragment separated using gel electrophoresis
  • southern blotting used to identify target sequence.