Biology

• What is recombinant DNA technology?

  Transfer of DNA fragments from one organism to another.
• Explain why transferred DNA can be translated within cells of recipient organisms?

  Genetic code in universal

  Transcription and translation mechanisms are universal
• Describe how DNA fragments can be produced using restriction enzymes.

  1. Restriction enzymes cut at specific base 'recognition sequences' either side of desired gene.

  2. Shape recognition site complementary to active site.

  3. Many cut in staggered fashion forming 'sticky ends' (single stranded stranded overhang)
• Describe how DNA fragments can be produced from mRNA?

  1. Isolate mRNA from a cell that readily synthesises protein coded for by desired gene.

  2. Mix mRNA with DNA nucleotides and reverse transcriptase - use mRNA as a template to synthesise a single strand of complementary DNA.

  3. DNA polymerase can form a second strand of DNA using cDNA as a template.
• Suggest two advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells.

  Much more mRNA in cells making proteins than DNA - easily extracted.

  In mRNA introns have been removed by splicing whereas DNA contains introns. - can be transcribed and translated by prokaryotes who cant remove introns by splicing.
• Describe how fragments of DNA can be produced by using the gene machine.

  1. Synthesises fragments of DNA quickly and accurately from scratch without the need for a DNA template - amino acid sequence determined allowing base sequence to be established.

  2. Do nit contain introns so can be transcribed and translated from prokaryotes.
• Name an in vitro and in vivo technique to amplify DNA fragments.

  In vitro - polymerase chain reaction.

  In vivo - culturing transformed host cells.
• Explain how DNA fragments can be amplified using PCR.

  1. Mixture heated to 95 degrees

  Allowing for separation of DNA strands, by breaking of hydrogen bonds.

  2. Mixture cooled to 55 degrees

  Allows primers to bind to DNA fragment template strand forming hydrogen bonds between complementary bases.

  3. Mixture heated to 72 degrees.

  Nucleotides align next to complementary exposed bases

  DNA polymerase joins adjacent nucleotides forming phosphodiester bonds.
• Explain the role of primers in DNA.

  Primers are short single stranded DNA fragments.

  Complementary to the DNA base sequence at start of desired gene.

  Allowing DNA polymerase to bind to start synthesis

  Two different primers are required (base sequences are different at both ends).
• Suggest why DNA replication eventually stops in PCR.

  There are a limited number of primers and nucleotides which are eventually used up.
• Summarise the stages in amplifying DNA in vivo.

  1. Add promoter and terminator regions to DNA fragments.

  2. Insert DNA fragments and marker genes into vectors using restriction enzymes and ligases.

  3. Transform host cells by inserting these vectors.

  4. Detect genetically modified transformed cells by identifying those containing marker genes.

  5. Culture these transformed host cells allowing them to divide and form clones.
• Explain why promoter regions are added to DNA fragments that are used to genetically modify organisms.

  Allow transcription to start by allowing RNA polymerase to bind to DNA.

  Can be selected to ensure gene expression happens in only specific cell types.
• Explain why terminator regions are added to DNA fragments that are used to genetically modify organisms.

  Ensure transcription stops at the end of a gene by stopping RNA polymerase.
• Explain the role of enzymes in inserting DNA fragments into vectors.

  1. Restriction endonuclease/ enzymes cut vector DNA - Same enzyme used to cut the gene out so vector DNA fragments have sticky ends that can join by complementary base pairing.

  2. DNA ligase joins DNA fragment to vector DNA - forming phosphodiester browns between adjacent nucleotides.
• Describe how host cells are transformed using vectors.

  Plasmids enter cells

  Viruses enter their DNA into cells which is then integrated into host DNA.
• Explain why marker genes are inserted into vectors.

  Allow for detection of genetically modified / transgenic organisms.

  As not all cells will take up vector and be transformed.
• Suggest how recombinant DNA technology is helpful in medicine.

  GM bacteria produce human protein- more ethically acceptable.

  GM animals / plans produce pharmaceuticals

  Gene therapy
• Suggest how recombinant DNA technology is helpful in agriculture.


  GM crops resistant to herbicides - only weeds killed when crops sprayed.

  GM crops resistant to insect attack - reduce use of insecticide

  GM crops have additional nutritional value.

  GM animals with increased growth hormone production.
• Suggest how recombinant DNA technology is helpful in industry.


  GM bacteria produce enzymes used in industrial processes.
• Describe gene therapy.


  Introduction of new DNA into ells often containing healthy and functional alleles

  To overcome effect of faulty / non functional allele in people with genetic disorders.
• Suggest some issues associated with gene therapy.

  Effect is short lived as modified cells have limited lifespan.

  Immune response against genetically modified cells or viruses due to antigen recognition.

  Long term effect is unknown - side effects may cause cancer
• Suggest why humanitarians support recombinant DNA technology.

  GM crops increase yield - increased global food production

  Gene therapy has potential to cure genetic disorders

  'Pharming' makes medicine more available and cheaper.
• Suggest why environmentalists are against recombinant DNA technology.

  Recombinant DNA technology may be passed to other plants.

  Potential effects on food webs

  Large biotech companies may control technology and own patents.
• What are DNA probes?


  Short single stranded pieces of DNA

  Base sequence is complementary to bases on part of target alleles.

  Usually labels with a fluorescent or radioactive tag.
• Suggest why DNA probes are longer than a few pieces.

  A sequence of few bases would occur at many places throughout the genome.

  Longer sequences are only likely to occur in target allele.
• What is DNA hybridisation?


  Binding of single stranded DNA probe to complementary single strand of DNA

  Forming hydrogen bonds / base pairs
• Explain how genetic screening can be used to locate specific alleles of genes.

  1. Extract DNA and amplify by PCR.

  2. Cut DNA at specific base sequences using restrictions enzymes.

  3. Separate DNA fragments using gel electrophoresis.

  4. Transfer to a nylon membrane and treat to form single strands with exposed bases.

  5. Add labelled DNA probes which hybridise / bind to target alleles.

  6. To show bound probe expose membrane to UV light if a fluorescently labelled probe was used.
• What is gel electrophoresis?


  A method used to separate nucleic aid fragments or proteins.

  According to length / mass and charge .
• Explain how gel electrophoresis can bee used to separate DNA fragments.

  1. DNA samples loaded into wells in a porous gel and covered in buffer solution

  2. Electrical current passed through - DNA negatively charged so moves towards positive electrode.

  3. Shorter DNA fragments travel faster so further.
• How can data showing results of gel electrophoresis be interpreted?

  Run a standard with DNA fragments / proteins of a known length.

  Compare to position of unknown DNA fragments/ proteins to estimate size.

  Shorter DNA fragments / proteins travel further.
• Describe examples of the use of labelled DNA probes.

  Screening patients for heritable conditions.

  Screening patients for drug responses

  Screening patient for health risks.
• Describe the role of a genetic counsellor.

  1. Explain results of genetic screening, including consequences of a disease

  2. Discuss treatments available for genetic condition

  3. Discuss lifestyle choices / precautions that might reduce risk of a genetic condition developing eg. regular screening for tumours or a mastectomy

  4. Explain probability of condition / alleles being passed onto offspring → enable patients to make informed decisions about having children
• What is personalised medicine?

  Medicine tailored to an individual's

  genotype / DNA

  Increasing effectiveness of treatment

  eg. by identifying the particular mutation / allele causing cancer and treating it with tailored drugs
• What are the advantages of genetic screening?

  Enable people to make lifestyle choices to reduce chances of developing disease.

  Allows people to make informed decision about having biological children.

  Allows use of personal medicine increasing effectiveness of treatment.
• What are the disadvantages of genetic screening?

  Screening for incurable disease can lead to depression.

  Could lead to discrimination by insurance companies

  May cause undue stress if patient doesn't develop disease.
• What are variable number tandem repeats?

  Repeating sequences fo nucleotides / bases

  Found within non-coding sections of DNA at many sites throughout an organisms genome.
• Why are VNTR's useful in genetic fingerprinting?

  Probability of two individuals having the same VNTRs is very low

  As an organism's genome contains many VNTRs and lengths at each loci differ between individuals
• Explain how genetic fingerprinting can be used to analyse DNA fragments.

  1. Extract DNA from sample (eg. blood cells) and amplify by PCR

  2. Cut DNA at specific base sequences / recognition sites (either side of VNTRs) using restriction enzymes

  3. Separate VNTR fragments according to length using gel electrophoresis (shorter ones travel further)

  4. Transfer to a nylon membrane and treat to form single strands with exposed bases

  5. Add labelled DNA probes which hybridise / bind with complementary VNTRs (& wash to remove

  unbound probe)

  6. To show bound probe, expose membrane to UV light if a fluorescently labelled probe was used
• Compare and contrast genetic fingerprinting with genetic screening.

  Both use PCR to amplify DNA sample

  Both use electrophoresis to separate DNA fragments

  Both use labelled DNA probes to visualise specific DNA fragments

  Genetic fingerprinting analyses VNTRs whereas genetic screening analyses specific alleles of a gene.
• Explain how genetic fingerprinting can be used to determine genetic relationships.

  More closely related organisms have more similar VNTRs, so more similarities in genetic fingerprints

  Paternity testing - father should share around 50% of VNTRs / bands with child (due to inheritance)