3.8.4 GENE TECHNOLOGIES

• Recombinant DNA Technology - Transferring a fragment of DNA from one organism to another (combining genetic material from different sources). 

  Genetic code universal, so are transcription and translation mechanisms - transferred DNA can be translated to produce protein in recipient cells. 

  • Recipient and donor organisms don’t have to be from same species. 
  • Transgenic organisms - Organisms containing transferred DNA.
• Recombinant DNA Technology 1 - Making DNA Fragments:

  • First need to get a DNA fragment containing gene to be transferred (target gene).
  • DNA fragment - bit of DNA containing a gene.

  3 Ways:

  • Using reverse transcriptase to convert mRNA to complementary DNA (cDNA)
  • Using restriction endonuclease enzymes to cut a fragment
  • Using a gene machine to create a gene
• Making DNA Fragments - Using reverse transcriptase to convert mRNA to complementary DNA (cDNA):

  mRNA easier to obtain than DNA containing target gene.  

  Used as templates to make lots of DNA.

  • mRNA isolated from cells, mixed with free DNA nucleotides and reverse transcriptase. The reverse transcriptase uses the mRNA as a template to synthesise new strands of cDNA. DNA produced is called complementary DNA (cDNA).
• Making DNA Fragments - Using restriction endonuclease enzymes to cut a fragment:

  Restriction endonucleases recognise specific palindromic sequences (complementary to active site) and cut the DNA at these places. 

  • If sequences present at either side of desired DNA fragment, endonucleases used to separate it - DNA sample incubated w specific endonuclease, which cuts DNA fragment out via hydrolysis.
  • Sometimes cut leaves sticky ends — small tails of unpaired bases at each end of the fragment. Can be used to anneal DNA fragment to another that has complementary sticky ends.
• Making DNA Fragments - Using a gene machine:

  DNA fragments synthesised from scratch. DNA sequence does not have to exist naturally.

  • Required sequence designed.
  • First nucleotide in sequence fixed to a support e.g. a bead.
  • Nucleotides added in correct order, as well as protecting groups. Protecting groups ensure nucleotides joined at right points, to prevent unwanted branching.
  • Short sections of DNA (oligonucleotides) produced, approx 20 nucleotides long. Once complete, broken off support and protecting groups removed. Oligonucleotides joined together to make longer DNA fragments.
• Recombinant DNA Technology 2 - Amplifying DNA Fragments:

  Gene cloning makes many identical copies of a gene.

  2 techniques:

  • In vivo cloning — gene copies made within living organism. As organism grows and divides, it replicates the DNA, creating multiple copies of the gene.
  • In vitro cloning — gene copies made outside of living organism using the polymerase chain reaction (PCR).
• In vivo cloning. Part 1 — Making recombinant DNA

  Insert DNA fragment into a vector’s DNA.

  • Vector DNA isolated.
  • Cut open using same restriction endonuclease used to isolate DNA fragment containing target gene. Means sticky ends of vector DNA complementary to those of DNA fragment containing gene.
  • Vector DNA and DNA fragment mixed together with DNA ligase, which joins the sticky ends of the DNA fragment to those of vector DNA. Process called ligation.
  • Recombinant DNA - New combination of bases in the DNA (vector DNA + DNA fragment).
• Vector - something that’s used to transfer DNA into a cell. Can be plasmids or bacteriophages (viruses that infect bacteria). 
• In vivo cloning. Part 2 — Transforming cells

  Vector with recombinant DNA used to transfer gene into cells. 

  Host cells that take up vectors = transformed. 

  If plasmid vector used, host cells have to be persuaded to take it in.

  • Eg. Host bacterial cells placed into ice-cold calcium chloride solution to make cell walls more permeable. Plasmids added and mixture heat-shocked (heated to around 42 °C for 1-2 minutes) - encourages cells to take in plasmids.
  • Bacteriophage vector - infects host bacterium by injecting its DNA. The phage DNA (with target gene in it) integrates into bacterial DNA.
• In vivo cloning. Part 3 — Identifying transformed cells

  Marker genes identify the GM cells:

  • Inserted into vectors at same time as target gene. Any transformed host cells will contain target gene + marker gene.
  • Transformed cells produce colonies where all cells contain cloned gene and marker gene.
  • Marker gene can code for antibiotic resistance (only transformed cells with marker gene survive and grow in certain antibiotic). Or code for fluorescence (only transformed cells fluoresce under UV light).
  • Identified transformed cells allowed to grow more, producing many copies of cloned gene.
• Promoter regions = DNA sequences that tell the enzyme RNA polymerase where to start producing mRNA. Terminator regions tell it where to stop.
• (In vivo cloning)

  To make transformed host cells produce protein coded for by the DNA fragment, need vector to contain specific promoter and terminator regions. 

  Without right promoter region, DNA fragment won’t be transcribed by host cell and protein won’t be made. 

  Promoter and terminator regions may be present in vector DNA or have to be added with fragment.
• In vitro cloning - Polymerase chain reaction.

  • Reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase. 
  • Heated to 95 °C to break the H bonds between 2 strands of DNA.
  • Cooled to 50-65 °C so primers can bind (anneal) to strands.
  • Heated to 72 °C, so DNA polymerase lines up free DNA nucleotides and joins nucleotides together. Specific base pairing - new complementary strands formed.
  • 1 cycle of PCR - 2 new copies of fragment of DNA formed. Cycle starts again with all 4 strands (2 original, 2 new) used as templates.

  Each PCR cycle doubles the amount of DNA.
• Primers - short pieces of DNA complementary to bases at start of desired fragment.
• Genetic Engineering - Microorganisms, plants and animals transformed using recombinant DNA technology. Transformed microorganisms can be made using same technology as in vivo cloning.
• • Transformed plants — a gene that codes for desirable protein inserted into a plasmid. Plasmid added to a bacterium (vector) to get gene into the plant cells. If the right promoter region has been added along with the gene, the transformed cells will be able to produce the desired protein.
• Transformed animals — a gene that codes for a desirable protein inserted into an early animal embryo or egg cells of a female. Early embryo - all body cells of resulting transformed animal will contain the gene. Egg cells - When female reproduces, all cells of her offspring will contain the gene.
• Transformed organisms - Promoter regions that are only activated in specific cell types can be used to control exactly which of an animal’s body cells the protein is produced in. If the protein is only produced in certain cells, it can be harvested more easily. Producing the protein in the wrong cells could also damage the organism.
• Recombinant DNA technology could be used to treat human diseases. (Gene therapy).

  Altering defective genes (mutated alleles) inside cells. 

  If disorder caused by:

  • 2 mutated recessive alleles - add a working dominant allele (supplement faulty alleles).
  • A mutated dominant allele - silence dominant allele (e.g. by sticking a bit of DNA in middle of allele so doesn’t work any more).

  Both involve inserting a DNA fragment into person’s original DNA. Requires a vector to get DNA into cell.
• Gene Therapy

  2 types:

  • Somatic therapy - Altering alleles in body cells, particularly cells most affected by disorder. Doesn’t affect sex cells (sperm or eggs), so any offspring could still inherit disease.
  • Germ line therapy - Altering alleles in sex cells. Every cell of any offspring produced from these cells will be affected by the gene therapy and they won’t suffer from disease. Currently illegal in humans.
• DNA Probes -

  • Used to locate specific alleles of genes/see if person’s DNA contains a mutated allele that causes a genetic disorder. 
  • Short strands of DNA with specific base sequence complementary to that of part of a target allele. 
  • DNA probe will bind (hybridise) to target allele if present in a DNA sample.
• • To produce DNA probe, first need to sequence allele screening for. Then use PCR to produce multiple complementary copies of part of the allele (the probes).
• DNA probe has a label attached, so can be detected. Radioactive label (detected using X-ray film) or fluorescent label (detected using UV light).

  Eg fluorescently labelled probes: 

  1. DNA sample digested into fragments using restriction enzymes and separated using electrophoresis.
  2. Separated DNA fragments transferred to a nylon membrane and incubated with a fluorescently labelled DNA probe. If allele is present, DNA probe will bind (hybridise) to it.
  3. Membrane exposed to UV light and if the gene is present, there will be a fluorescent band.
• Screening with DNA Probes -

  DNA microarray (glass slide with microscopic spots of different DNA probes attached to it in rows) used to screen for lots of different genes at same time. 

  • Sample of fluorescently labelled human DNA washed over array. If DNA contains any sequences that match any probes, it will stick to array. 
  • Array washed, to remove any unstuck fluorescently labelled DNA
  • Visualised under UV light. Any labelled DNA attached to a probe will show up (fluoresce). Any spot that fluoresces means person’s DNA contains that specific allele. 
• Screening Results used in:

  • Genetic counselling (advising on risks of genetic disorders, prevention, treatment etc)
  • Screening helps identify if someone is a carrier of mutated allele.
  • Personalised medicine - Genes determine how body responds to certain drugs - certain drugs more effective for some than others. Doctors use genetic info to predict response to different drugs and only prescribe most effective ones.
• Some of genome consists of variable number tandem repeats (VNTRs) — base sequences that don’t code for proteins and repeat next to each other multiple times.

  • Number of times these sequences are repeated differs from person to person (so also the length of these sequences in nucleotides).

  Number of times a sequence repeated (and so number of nucleotides) at different places in genome can be compared between individuals = genetic fingerprint. Probability of 2 individuals having same genetic fingerprint (same VNTRs) very low.
• Producing Genetic Fingerprints (Gel Electrophoresis)

  Step 1 — PCR used to make DNA fragments:

  • Sample of DNA obtained. 
  • PCR used to clone DNA fragments containing the VNTRs. 
  • Primers bind to either side of repeats so whole repeat amplified (copied many times). Different primers used for each position under investigation. 
  • End up with DNA fragments where length (in nucleotides) corresponds to number of repeats person has at each specific position. 
  • Fluorescent tag added to all DNA fragments (usually to primers) so can be viewed under UV light.
• Producing Genetic Fingerprints (Gel Electrophoresis)

  Step 2 — Separation of the DNA fragments:

  • DNA mixture placed into well in gel and covered in buffer solution that conducts electricity. 
  • Electrical current passed through gel — DNA fragments negatively charged so move towards positive electrode at the far end of gel. 
  • Shorter DNA fragments move faster and travel further through gel, so DNA fragments separate according to length. Produces pattern of bands.
• Producing Genetic Fingerprints (Gel Electrophoresis)

  Step 3 — Analysis

  Gel placed under UV light. DNA fragments seen as bands, making up the genetic fingerprint. 

  • A DNA ladder can be added to one well (a mixture of DNA fragments of known length) - allows you to work out length of other bands on gel. 

  2 genetic fingerprints can be compared, e.g. if both fingerprints have a band at the same location on the gel it means they have the same number of nucleotides and so the same number of VNTRs at that place.
• Uses of Genetic Fingerprinting

  • Determining genetic relationships - More bands on a genetic fingerprint that match, the more closely related (genetically similar) 2 people are.
  • Determining genetic variability within a population - Compare repeats in genome to find out how genetically varied.
  • Forensic science - Compare genetic fingerprints produced from DNA samples from crime scenes and suspects.
  • Medical Diagnosis - diagnose genetic disorders and cancer.
  • Animal and plant breeding - prevent inbreeding (fingerprinting used to identify how closely related individuals are)