7.1 using gene sequencing

Cards (13)

  • 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.