Application of reproduction and genetics

• Human Genome Project


  Began in 1990, took 10 years to complete, analysis of sequences took much longer
• Main aims of the Human Genome Project

  • Identify all the genes in the human genome and their loci
  • Determine the sequence of the 3.6 billion bases in the human genome and store in databases
  • Consider the ethical, social and legal issues that arise from storing this information
• The number of genes present in the human genome is around 20,500
• There are large numbers of repeating sequences called STRs (short tandem repeats)
• Sanger sequencing


  Method of sequencing used in the Human Genome Project, named after the scientist who invented it
• Sanger sequencing process


  1. Sequence small DNA fragments around 800 bases long created by restriction enzymes

  2. DNA polymerase used to synthesise complementary strands using PCR

  3. Four reactions carried out (one for each nucleotide) with some stop nucleotides incorporated

  4. Resulting DNA fragments of different lengths run on agarose gel and sequence determined from banding pattern
• Sanger sequencing is very slow, taking days to accurately sequence thousands of bases
• Next Generation Sequencing (NGS)


  Can sequence entire genomes in hours
• The 100K Genome Project launched in 2012 using NGS to sequence 100,000 genomes from healthy individuals and patients to identify genetic variances
• Ethical concerns with genome projects

  • Passing genetic predisposition information to insurance companies
  • Using ancestral relationships for social discrimination
  • When to inform people of genetic predispositions like Alzheimer's
  • Screening embryos for desirable traits
  • Ensuring safe storage of patient data
• The mosquito Anopheles gambiae responsible for malaria transmission has had its genome sequenced
• In 2015, gene-editing technology was used to produce a genetically modified mosquito that could produce antibodies to the malaria parasite
• Polymerase Chain Reaction (PCR)


  Technique that produces a large number of copies of specific fragments of DNA rapidly
• Requirements for PCR


  • Heat stable DNA polymerase
  • Short single-stranded DNA primers
  • Deoxyribonucleotides
  • Buffer
• PCR process


  1. Heat to 95°C to separate DNA strands

  2. Cool to 50-60°C to allow primers to anneal

  3. Heat to 70°C for DNA polymerase to extend complementary strands

  4. Repeat 30-40 times
• PCR has limitations including amplifying any contamination and sometimes incorporating incorrect nucleotides
• Short Tandem Repeats (STRs)


  Variable regions of non-coding DNA used for genetic fingerprinting
• An example STR is D7S280 on chromosome 7 which has 6-15 repeats of the gata sequence
• Genetic fingerprinting


  Using PCR to amplify specific STR sequences and visualise the unique banding pattern on a gel to identify an individual
• DNA probes


  Short pieces of single-stranded DNA labelled with a radioactive or fluorescent marker to detect complementary sequences
• DNA profiling is a non-invasive procedure using hair or mouth swab samples
• Probe


  Short piece of DNA that is labeled with a beacon of radioactive marker and to detect the presence of a specific base sequence in another piece of DNA, by complementary pairing
• DNA hybridisation


  1. DNA fragment which contains the sequence of interest is identified by its fluorescence or radioactive signal

  2. DNA from the gel is transferred to a nylon membrane

  3. Membrane is exposed to X-ray film producing an autoradiograph
• DNA profiling


  • Non-invasive procedure requiring hair samples or mouth swab to collect DNA
  • DNA can be further amplified by PCR
• Uses of DNA profiling

  • Provide forensic evidence to identify or rule out suspects
  • Prove paternity or maternity
  • Immigration applications
  • Phylogenetic studies
• DNA profiling cannot guarantee a match: at best a genetic fingerprint has a 1 in 1 billion chance that someone else could have the same profile
• Ethical and legal concerns exist over the storage and use of DNA profiles by agencies such as the police, or health insurance providers
• DNA evidence in criminal cases is often relied upon too much to prove guilt, instead of supporting other evidence
• A DNA sample from a crime scene may strongly indicate that a particular individual was present, not that they necessarily committed the crime
• Recombinant DNA


  DNA produced by bringing together genetic material from two different sources
• Transgenic


  An organism that has been genetically modified by the addition of a gene from another organism
• Restriction enzymes


  Bacterial enzymes that cut up any foreign DNA which enters a cell
• Using restriction enzymes to insert a gene into a plasmid

  1. Cut plasmid with restriction enzyme

  2. Cut foreign DNA with same restriction enzyme

  3. Insert DNA using DNA ligase
• DNA ligase


  Bacterial enzyme that joins the sugar-phosphate backbones of two pieces of DNA together
• The second marker gene in the plasmid is used to confirm insertion of the target gene
• Reverse transcriptase


  Enzyme that produces complementary or copy DNA (cDNA) from an mRNA template
• Using reverse transcriptase to produce human insulin

  1. Extract mature miRNA coding for insulin from pancreas

  2. Reverse transcribe miRNA to cDNA

  3. Replace human regulator sequence with bacterial regulator

  4. Insert cDNA into plasmid using restriction enzymes and ligase
• Prokaryotes don't have introns in their DNA so this procedure gives intron-free DNA for expression
• Advantages of genetically engineering bacteria

  • Allows production of complex proteins/peptides
  • Production of medicinal products like human insulin
  • Can be used to enhance crop growth
  • Used to treat tooth decay
• Disadvantages of genetically engineering bacteria

  • Technically complicated and expensive
  • Difficulties identifying genes of value
  • Synthesis of required protein may involve multiple genes
  • Not all eukaryote genes will express in prokaryote cells