7. Modern genetics

• What is a genome?

  All the genetic material in a single somatic cell from an orgnaism
• When is DNA amplified?

  When a large number of identical copies are made from it
• What does gene sequencing involve?

  Finding the nucleotide base sequence of a cell genome
• What is a polymerase chain reaction?

  The process used to make a large amount of identical DNA from a small sample
• What is a polymerase chain reaction similar to?

  Semi-conservative replication
• Where is the polymer chain reaction carried out?

  In automated thermal cyclers
• What four things are within a thermal recycler?

  Template DNA, DNA nucleartides, thermostable DNA polymerase and primers
• Within the thermal recycler what is a template DNA for?

  The DNA to be amplified
• What does thermostable DNA polymerase do?

  The enzyme that catalyses the formation of phosphodiester bond between DNA nucleotides
• What is a primer?

  A single stranded oligopoly nucleotide that allows DNA polymerase to begin replication of a template strand of DNA
• Why is electrophoresis used?

  To separate charge substances by causing them to migrate through a porous medium placed in an electrical field
• What is the first stage of electrophoresis?

  A sample of DNA is placed in walls at one end of an agarose gel
• What is the second stage of electrophoresis?

  A power supply creates an electrical field along the gel with the walls at a negative poll
• What is the third stage of electrophoresis?

  The charged fragments migrate from the negative to the positive poll
• What is the fourth stage of electrophoresis?

  Paused in the gel allows small fragments to move faster than larger fragments
• What is the fifth stage of electrophoresis?

  The gel is fragile, so an imprint is made onto a more robust nylon membrane
• What are probes?

  Short single strands of DNA labelled with radioactive phosphorus
• What's the last stage of electrophoresis?

  An x-ray film is used as a permanent record of the results of electrophoresis
• What is the first step in PCR?

  Denaturing the template DNA in 95°C
• What is the second step in PCR?

  Annealing of the oligonucleotide primers at 50°C to 60°C
• What is the third step of PCR?

  Synthesis of new DNA at 74°C
• What's the fourth step of PCR?

  Repeat the cycle 25 to 30 times
• What is the difference with PCR when chain termination uses it?

  An abnormal nucleotide is used called dideoxynucleotides
• What prevents deoxyribose from forming a phosphodiester bond?

  Having a hydrogen atom on the third carbon not a hydroxyl group
• What causes DNA polymerase to stop?

  When a dideoxynucleotides is randomly incorporated into developing DNA chain
• What is the first step of the chain termination sequence technique?

  Add a copy of the single strand DNA to be sequenced
• What is the second stage of chain termination sequence technique?

  Add DNA polymer
• What is the third stage of chain termination sequence technique?

  Add primers
• What is the fourth stage of chain termination sequence technique?

  Add molecules of all four normal DNA nucleotides
• What is the fifth stage of chain termination sequence technique?

  Add molecules of only one type of deoxy nucleotides carrying, A,G,C,or T
• What is the sixth stage of chain termination sequence technique?

  Use gel electrophoresis in order to separate the fragments
• When using electrophoresis in chain termination, what can you infer about the fragment that moves the furthest?

  That the base that is in the sequence it at the front so the complimentary base will be at the front of the template strand
• Can gene sequencing be automated?

  Yes
• In automated gene sequencing what happens?

  One reaction tube is used, 4 types of dideoxynucleotides are dyed, electrophoresis occurs inside a gel within a capillary tube, coloured bands are read, base sequence of different fragments of DNA are pieced together to give the base sequence of the template DNA
• In chain termination, what is the first step to completing a gene sequence?

  Find overlaps in the base sequences of the DNA fragments
• In chain termination, what is the second step to completing a gene sequence?

  Arrange the overlaps in the base to find which fragments overrun an adjacent fragment
• In chain termination, what is the third step to completing a gene sequence?

  Arrange all the overlapping fragments into a consensus sequence, i.e one that all scientists in the field agree is an authentic representation of the sequence of each DNA molecule in the genome
• How is whole genome sequencing done?

  On the computer
• How do you use the result of the whole genome sequencing?

  Predict amino acid sequences of the polypeptides encoded by each gene, find the base sequence of the allele of a gene that results in a medical condition, find similarities in the base sequence of common genes to work out evolutionary relationships between different organisms
• What is a short tender repeat? (STR)

  Are sections of DNA with a repeated sequence of nucleotide bases