exam qs

• It is important that scientists report the results from failed attempts to produce transgenic animals. Why?

  • Scientists don't repeat mistakes
  • Saves time and money
• Suggest reasons why very few live births result from the many embryos that are implanted.

  • Foreign embryo is attacked by antibodies
  • DNA may be damaged
  • DNA may interfere with proteins
• When manufacturing large quanitites of insulin, why is it better to start by isolating mRNA from pancreas cells rather than DNA from pancreas cells?

  • mRNA doesn't have introns
  • larger amount of mRNA compared to DNA
  • Specific mRNA found in pancreas cells
• Other than restriction endonuclease, which enzyme must the scientist have added to the mixture to form recombinant plasmids?

  DNA ligase to bind the sticky ends and create phosphate backbone framework.
• Explain why pieces of DNA can join to the cut DNA of plasmids.

  • Sticky ends
  • Complementary bases
• Explain the purpose of attaching a fluorescent gene.

  • Acts as a marker gene
  • Identifies which cells have taken up the gene
  • Cells which glow have taken up the gene
• Explain ways in which the PCR differs from DNA replication in a cell.

  • PCR uses heat to separate strands
  • PCR replicates pieces of DNA because DNA has been cut
  • Primer added in PCR to initiate replication
• Explain why it takes longer to obtain a genetic fingerprint if the sample is small.

  PCR must be done to copy fragments of DNA.
• Suggest why scientists use marker genes

  • Not all cells take up the plasmid
  • Allows identification of cells that have taken up the plasmid
• Describe how copies of a gene can be inserted into plasmids.

  • Use same restriction endonuclease on plasmid that was used to obtain copies of gene
  • Same sticky ends are present on gene and plasmid
  • Add ligase to join complementary ends
• Explain what is meant by a vector (1)

  Carrier of DNA / gene into another cell
• Molecular biologists often use plasmids which contain antibiotic resistance genes.

  Explain the reason for this.

  • acts as marker gene
  • allows detection of cells containing plasmid / DNA
• Why are DNA primers added during PCR?

  • to mark beginning and / or ends of part of DNA needed
  • for attachment of enzymes or nucleotides
  • keeps strands apart
• What is the advantage of the enzyme used in PCR being thermostable?

  • would not be denatured
  • must withstand high temperatures
• Suggest why two different primers are required in PCR

  • base sequences at the ends of target sequence are different
  • one is at the beginning and one at the end
• After one cycle of PCR, there will be two copies of DNA; after two cycles, four copies and this doubling continues.
• Gel electrophoresis separates DNA fragments by their charge and size.
• Explain why radioactive DNA probes are used to locate specific DNA fragments

  • DNA invisible on gel
  • allows detection
• Promoter and terminator regions are added to fragments of DNA to ensure transcription is initiated and finished, so the gene can be expressed.
• Cut plasmids and lengths of foreign DNA can join. What features of their ends allow them to join?

  • unpaired bases / sticky ends
  • complementary base pairing
• Suggest one use of PCR

  • replication of DNA from crime scene
  • for DNA sequencing / gene cloning
• Give two ways in which PCR differs from the process of transcription.

  • transcription uses RNA polymerase
  • RNA nucleotides / uracil
  • one template strand in transcription / PCR both strands
  • start / stop codons
• Explain why the DNA fragments move different distances in the gel

  different lengths / sizes / masses
• Describe the role of restriction endonucleases in the formation os plasmids that contain donor DNA

  • cut open plasmid
  • cut donor DNA, to remove gene
  • cut donor DNA and plasmid with the same enzyme
  • sticky ends
  • pairing of complementary strand
• Describe the role of DNA ligase in the production os plasmids containing donor DNA

  • backbones joined / phosphodiester bonds
• Use your knowledge of enzymes to explain why restriction enzymes only cut DNA at specific restriction sites

  • Different lengths of DNA have different base sequences / cut at specific sequence
  • Results in different shape / different shape of active site
  • Therefore specific sequence will only fit active site of enzyme
• What is recombinant DNA

  • contain genes / sections of DNA
  • DNA from two species
• What is a gene probe?

  • strand of DNA
  • short strand
  • with base sequences at complementary to part of target gene
  • radioactive labelling / fluorescent labelling
• Describe how bacteria may be produced which may have the resistant gene in their plasmids (6)

  • make artificial DNA with correct sequences of bases
  • using DNA polymerase
  • cut plasmid open
  • with (same) restriction endonuclease / restriction enzyme
  • sticky ends / unpaired bases attached
  • use (DNA) ligase to join
  • return plasmid to (bacterial) cells
  • use of Ca2+ / calcium salts / electric shock
• Explain how the strands of DNA are separated during PCR (2)

  • by heating
  • to break hydrogen bonds between complementary bases
• Describe how an isolated gene can be replicated by PCR (4)

  • heat DNA to 90-95 degrees
  • H-bonds broken, strands separate
  • add primers
  • and nucleotides
  • cool so primers bind to DNA
  • DNA polymerase forms new strands / joins nucleotides
• Suggest one reason why DNA replication stops in PCR (1)

  • limited number of nucleotides
• Determining the genome of the viruses could allow scientists to develop a vaccine.

  Explain how. (2)

  • can identify proteins derived from genome
  • identify antigens for vaccine
• Describe the roles of two named types of enzymes used to insert DNA fragments into plasmids. (2)

  • Restriction (endonuclease) to cut plasmid / vector
  • Ligase joins gene / DNA to plasmid / vector
• Suggest two features of the structure of different proteins that enable them to be separated by gel electrophoresis (2)

  • mass / number of amino acids / polypeptides
  • charge
  • R groups
• Give two reasons why bacteria are able to use human DNA to produce human proteins (2)

  • genetic code is universal
  • mechanism of transcription is universal
  • mechanism of translation is universal
• Suggest and explain one reason why bacteria might not be able to produce every human protein (1)

  • Cannot splice (pre-mRNA), so cannot remove introns
  • Do not have Golgi (apparatus), so cannot process/modify (proteins)
  • Do not have transcriptional factor required so cannot carry out transcription / produce mRNA