Chapter 21

• What is DNA fingerpringintg?

  A method showing differences between individuals' DNA
• Where can we get samples of DNA?

  Blood, Semen, Saliva, Hair follicles etc...
• Why are there differences in DNA?


  Restriction endonuclease digests double stranded DNA and will recognise specific sequences, causing different sixes and lengths of DNA to be made.
• What is electrophoresis?


  Technique which separates DNA fragments by utilising their length and charge.
• What is the gel electrophoresis process?

  Samples are put into a gel medium and an electric current is passed through.

  The DNA, as it is negatively charged, moves towards the positive electrode.

  The smaller fragments move faster and therefore further in the same amount of time.
• What are DNA probes?

  Short, single-stranded DNA sequences used to detect complementary sequences in a sample.
• What will the DNA probes have for identification?


  Either a radioactivr tag or a fluorescent tag
• What are DNA probes used alongside?


  Southern blotting or electrophoresis
• How does a DNA probe work?


  Probe is mixed with the DNA of interest and hybridisation will occur between the probe and the target DNA.

  Positive results can be seen by exposing x-ray film.
• What is the whole process of genetic fingerprinting?

  1. Extraction
  2. Digestion
  3. Electrophoresis
  4. Transfer DNA to a nylon membrane.
  5. Hybridise with gene probe.
  6. Develop using x-ray film
• What is the use of Genetic fingerprinting?


  Paternity cases, forensics, medical diagnosis, plant and animal breeding.
• What is the polymerase chain reaction?

  A method of copying fragments of DNA through a automated and rapid proccess.
• What are the required components of PCR?


  DNA fragment, DNA polymerase, Primers, Nucleotides and a Thermocycler
• What are the main stages of PCR?


  Separation of DNA strand, Addition of the primers and Synthesis of the DNA
• What occurs in the separation of the DNA strand in PCR?


  Temp in thermocycler reaches 95 degrees Celsius.

  Breaks the H bonds between the strandes.
• What happens in the addition of the primers stage of PCR?


  Thermocycler cooled to 55 degrees, so the primers can join to their complementary DNA strands and at the end of the fragments.

  This provides starting points for the DNA polymerase.

  This also prevents strands from rejoining.
• What happens in the synthesis stage of PCR?


  Temp increased to 72 degrees, which is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands.

  Begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.
• What is the result of PCR?


  Because both separated strands are copied simultaneously there are now two copies of the original fragment.

  Process can then repeat, resulting in 4 strands, the whole cycle takes about 2 minutes and so a 100 billion copies can be done in just a few hours.
• Polymerase chain reaction is known as what type of cloning?

  In vivo cloning
• What is restriction mapping?


  Cutting DNA with a series of different restriction endonucleases, separating them by electrophoresis and then working out distances between recognition sites using the fragments produced
• What is the first step for the Use of vectors in in vivo cloning:

  DNA fragment is cut using restriction enzymes to make complementary 'sticky ends'.

  Plasmid is cut at the same restriction sites to make the same complementary ends.

  Combined to form a genetically engineered plasmid using DNA ligase to form phosphodiester bonds.
• What must you add if you want a specific gene to be expressed in a new host?


  Promoter region and terminator
• What is the second step for the use of vectors in in vivo cloning?


  Gene inactivation:

  Post transformation grow the bacteria on two mediums with different antibiotics.

  Evaluate which bacteria live and die.

  Use this to work out which resistance gene was cut into two by the restriction site location.
• What are the tools needed for recombinant DNA technology?


  Restriction endonucleases, DNA ligase, Reverse transcriptase.

  Vectors.

  New hosts.
• The enzymes of recombinant DNA technology?


  1. Restriction endonucleases = Hydrolyse double stranded DNA within the molecule and recognise specific base sequences called restriction sites
  2. DNA ligases = join double stranded DNA fragments, as the ends must be complementary. Condensation reaction with phosphodiester bonds being formed.
  3. Reverse transcriptase = makes a double stranded DNA copy of an mRNA chain. Found in retroviruses.
• The vectors in recombinant DNA technology are usually?

  Plasmids that carry a gene of interest
• What are the common new hosts for recombinant DNA technology?


  Usually bacterium as you get lots of copies.

  Sometimes viruses, but you get less copies.
• What is transformation?


  DNA from one organisms into another (uses heat shock of electricity)
• How do you produced DNA fragments?


  Restriction enzymes that create sticky ends.

  Reverse transcriptase.

  The Gene machine which makes lengths of double stranded DNA of required base sequences using PCR and Vectors.
• What are some of the benefits of DNA technology?


  Medical advancements, forensic identification, agriculture improvements.
• What are some of the risks of DNA technology?


  Overuse, Pathogenic resistance, Ecological consequences of genetically engineered organisms is unknown
• What is genetic screening?

  The process of analysing an individual's DNA to identify genetic variations or mutations that may be associated with certain diseases or conditions.

  You must screen for both dominant allele and recessive as otherwise you wont be able to tell if they are homozygous or heterozygous.
• What is personalised medicine?

  Screening for predisposition of certain diseases and adjusting the lifestyle accordingly.
• What is genetic counselling?


  Form of social work where advice and info are given out to enable people to make personal decision about themselves or their offspring.

  Have to research family history of an inheritable disease and advise parents of the likelihood of it impacting them.