Gene technologies

• what are DNA probes and what are the two types ?

  A dna probe is a short, single stranded length of DNA that has a known base sequence complementary to the specific of a known allele.

  • radioactively labelled probes - which are made of nucleotides with the isotope 32P. Which can be identified using an X-ray film
  • fluorescently labelled probes- which emit light (fluoresce) under certain conditions. For instance when the probe has bounds to the target DNA sequence.
• What are DNA probes used for ?

  Use to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks.
• how do DNA probes work ?

  • a DNA probe is made that has base sequences that are complementary to part of the base sequence of the DNA that makes up the allele of the gene that we want to find.
  • the patients DNA sample is treated to make it single stranded and it is then mixed with the DNA probes.
  • the DNA probes then bind to complementary base sequence on the strands. this is known as DNA hybridisation.
• describe DNA hybridisation

  • the patients DNA sample is heated to make it single-stranded
  • the heat causes the hydrogen bonds between bases to break (denaturing)
  • the patients single stranded DNA sample is mixed with the DNA probe and cooled.
  • any complementary sequences can align and form hydrogen bonds (annealing)
  • some of the patients DNA samples will anneal back together but some will anneal with the DNA probe.
• how do we create the DNA probe ?

  1. we use DNA sequencing methods to determine the the sequence of nucleotide bases of the mutants allele we are trying to locate.
  2. a fragment of dna is produced that has a sequence of bases that are complementary to the mutant allele.
  3. multiple copes of our DNA probe is made by using polymerase chain reaction
  4. then either a fluorescent dye is added or the isotope 32P is added.
• genetic screening

  Genetic screening is the testing of an embryo, fetus or adult to analyse the DNA.
• what are variable number tandem repeats (VNTRs)

  regions found in the non-coding part of DNA. For every individual the number and length of VNTRs has a unique pattern.
• extraction (genetic fingerprinting)

  the smallest sample of DNA can be collected for genetic fingerprinting . This can be from blood, body cells or hair follicles. The sample size can be increased using PCR
• Digestion (genetic fingerprinting)

  Restriction endonucleases are added to cut the DNA into smaller fragments. (enzymes are chosen which cut close to the target VNTRs)
• Seperation (genetic fingerprinting)

  • DNA samples are loaded into small wells in agar gel.
  • the gel is placed in a buffer liquid with an electrical voltage applied.
  • DNA is negatively charged so the DNA samples move through the gel towards the positive end of the gel.
  • The agar gel creates resistance for the moving DNA and smaller pieces of DNA move faster and further along the gel.
  • An alkaline is then added to separate the double strands of DNA.
• hybridisation(genetic fingerprinting )

  • radioactive or fluorescent DNA probes are now used to bind with VNTRs
  • different DNA probes are mixed with the single stranded DNA VNTRs on the agar gel for them to bind (hybridise)
• development (genetic fingerprinting)

  • the agar gel will shrink and crack as it dries and therefore the VNTRs and DNA probes are transferred to a nylon sheet.
  • the nylon sheet is then exposed to X-rays to visualise the position of the radioactive gene probes. or UV light if fluorescent probes where used
• analysis

  the position of the DNA bands are compared to identify the genetic relationships, the presences of the diseas causing gene and to match unknown sample from a crime scene.
• what are the steps of genetic fingerprinting ?

  1. Extraction
  2. digestion
  3. seperation
  4. hybridisation
  5. development
  6. analysis
• What is recombinant DNA technology ?

  It is a form of genetic engineering that involves the transfer of fragments of DNA from one organism into another organism.
• what is recombinant DNA ?

  The combination of dna from two different organisms
• what is a transgenic organism ?

  A genetically modified organism that contains recombinant dna
• How is it that DNA of one organism is accepted by a different species ? 


  The genetic code is universal. The same codon codes for the same amino acid in all living organism. The mechanisms for Transcription and translation are also universal.
• what are the stages of using recombinant dna ?

  1. isolation - of the dna fragment that have the gene for the desired protein.
  2. insertion - of the dna fragment into a vector
  3. transformation - transfer of dna into a suitable host
  4. identification - of the host cells that have succesfully taken up the gene by using gene markers
  5. growth/cloning - of the population of host cells
• what the different ways of isolating a DNA fragment ?

  Reverse transcriptase

  restriction endonucleases

  gene machine
• explain how reverse transcriptase is used to create a DNA fragment

  Make DNA copies from mRNA

  naturally occur in retroviruses (HIV)

  1. A cell that readily produces the protein is selected (the B-cells of the islets of langerhans from the pancrease used to produce insulin)
  2. these cells have large quantitities of the relevant mRNA
  3. the reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA Sequence.
  4. Single stranded DNA is made (cDNA)
  5. DNA polymerase makes the cDNA double-stranded
  6. the cDNA doesn’t have introns
• explain how restriction endonucleases are used in producing DNA fragments

  enzymes that cut DNA at restriction sites to form DNA fragments. there are two ways in which the enzymes cut DNA:

  • produces blunt ends- some cut through the same location in the double stranded DNA to produce a blunt end
  • produce staggered ends(sticky ends) - these ends are palindromic and are called sticky ends because they can join to complementary DNA base pairs
• explain how gene machines create a DNA fragment

  with knowledge of the genetic code scientist use computers to generate the nucleotide sequence to produce the gene.

  1. scientist identify amino acid sequence of the protein of interest, and the mRNA + DNA sequence from that
  2. DNA sequence is entered into a computer which has to pass biosecurity checks
  3. computer creates a small section of overlapping DNA strands called oligonucleotides.
• how can the specificity of restriction enzymes be investigated ?

  the specificity of restriction enzymes can be investigated using extracted DNA and gel electrophoresis.
• what is gel electrophoresis ?

  how does the separation of DNA occur ?

  a technique that is used to analyse DNA. it uses electric currents to separate DNA molecules according their size, made and charge.

  DNA is negatively charged due to the phosphate groups and so when placed in an electric field the molecules move(migrate) towards the positive electrode. the smaller molecules movequicker than the larger molecules.
• visualisation of restriction fragments


  as the restriction fragments move through the gel, bands of restriction fragments are formed.

  • it is stained which results in a series of coloured bands in the gel.
  • This could also be done by reading the DNA with radioactive marker or adding fluorescent probes.
• What are the two ways in which DNA fragments are cloned

  in vivo cloning

  in vitro cloning
• preparing the DNA fragment for insertion (in vivo cloning)

  promoter region must be added - this is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur.

  terminator must be added - this is added at the end of a gene. it causes RNA polymerase to detach and stop transcription. so only one gene at a time is copied into mRNA
• insertion of DNA into a vector 


  plasmids are used as a vector.

  1. the plasmid is cut open by the same restriction endonuclease (used to form DNA fragment)
  2. this creates the same sticky end
  3. therefore the DNA fragment sticky ends are complementary to the sticky ends on the plasmid.
  4. enzyme ligase then sticks them together(annealing)
  5. ligament catalyses the condensation reaction to form phosphodiester bonds between nucleotides.
• what is the ligase enzyme used for ?

  used to join DNA fragment and (cut) plasmid together. (annealing)

  catalyses the condensation reaction to form phosphodiester bonds between nucleotides.
• explain transformation

  the vector is inserted into the host cell. where the gene will be expressed to create the protein required.

  in order to do this the cell membrane of the hosT cell must be more permeable.

  to increase the permeability, the host cels are mixed with ca2+ and heat shocked (sudden increase in temperature)
• why wont all of the host cells posses the DNA fragments with the desired gene.

  -only a few bacterial cells host cells) take up the plasmids when the two are mixed together.

  -some of the plasmids will have closed up again without incorporating the DNA fragment.

  -the DNA fragment ends join together to form its own plasmid
• what are the three different methods of using marker gene to identify whether a gene has been taken up by bacterial cell?

  three different marker genes used are…

  1. antibiotic resistance gene
  2. genes coding for fluorescent proteins
  3. genes coding for enzymes
• how do you prepare plasmid For antibiotic-resistance marker gene

  1. Two genes inserted into plasmid

  2. Gene for resistance to tetracycline antibiotic

  3. Resistance to ampicillin gene

  4. DNA fragment inserted into middle of tetracycline gene - this gene will no longer create functional protein
• Identifying bacteria with plasmids

  1. Grow bacteria on agar

  2. Transfer bacteria colonies to plate with ampicillin antibiotic in agar - this shows you the bacteria that contain the plasmids with resistance ampicillin gene

  3. Transfer bacteria colonies to plate with tetracycline antibiotic in agar - those that grew means they don't contain the DNA fragment as they are resistant
• gel electrophoresis seperates out substances based of…

  Mass

  length

  charge
• what are primers 


  Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.
• Stages of PCR


  1. Separation of DNA strands

  2. Addition (annealing) of the primers

  3. Synthesis of DNA
• Separation of DNA strands


  Temperature is increased to 95 degrees Celsius, causing the two strands to separate (breaking the H-bonds)
• Addition (annealing) of the primers


  It is cooled to 55 degrees Celsius, causing the primers to anneal to their complementary base pairs at the end of the DNA fragment. The primers provide the starting sequence for DNA polymerase to begin DNA copying. Primers also prevent the two strands from rejoining.