6.3 Manipulating genomes

• What is DNA sequencing?

  Technique that allows genes to be isolated and read

  1. Compare individuals
  2. identify alleles that are only present in certain ones.
  3. Bioinformatics to link the gene to the phenotype/disease e
• Timeline of gene discovery

  1970 - structure of DNA was known

  1972 - gene that coded for a protein was sequenced

  1975 - whole genomes were sequenced
• Describe Sanger's Chain Sequencing Approach

  1. single stranded DNA used as a template
  2. 4 different dishes with adenine, thymine, guanine and cytosine and DNA polymerase
  3. Modified versions of 1 base is added (no more nucleotides can be added after it, truncated sequence
  4. Lots of different fragments produced and then they are pass through gel electrophoresis
  5. Shortest fragments travel the furthers
• Advantages and Disadvantages of Sanger's Method

  1. Safe and efficient
  2. DNA can be identified after gel electrophoresis using fluorescent dye and visible stain
  3. However, you have to count it one by one so its costly and time consuming
• Pyrosequencing

  1. DNA cut and degraded (using heat) into a single stranded template and the immobilised
  2. In the boiling tube - primer, enzymes (ATP sulfurylase, DNA polymerase, luciferase, apyrase) substrates (Luciferin, APS) one activated nucleotide
  3. activated molecule causes 2 phosphate to be released as pyrophosphate. combines with APS and ATP sulfuryase converted into ATP.
  4. Luciferase converts luciferin into oxyluciferin, AMP and light
  5. Light is detected and is proportional to number of adjacent activated nucleotides
• Advantages of pyrosequencing

  1. Processes millions of fragments rather than a few
  2. no need for gel electrophoresis
  3. better suited for short-read sequencing
• PCR

  biomedical technology in molecular biology that can amplify a short length of DNA into millions of copies
• What does PCR rely on

  PCR - Polymerase chain reaction

  1. DNA made of 2 anti-parallel backbone strands
  2. 5' end and 3' end, with DNA that only grows from the 3' end
  3. base pairs that line up accordingly
• How is PCR different from DNA replication

  Involves the replication of short sequences not whole chromosomes. Needs a DNA primer to provide a starting point for the DNA polymerase to bind to. Involves heating and cooling
• Heating and cooling (PCR)

  1. Denaturation - 95 degrees - separates DNA strands
  2. Annealing - 68 degrees - causes primers to bind
  3. Elongation - 72 degrees - DNA is replicated
• PCR Steps

  1. Cyclic reaction
  2. DNA mixed with nucleotides, Mg2+, primers and Taq polymerase
  3. heated - 95 degrees - breaks the hydrogen bonds between complementary base pairs. Double strand denatures into single strand
  4. cooled - 68 degrees - primers can anneal - bind by hydrogen bonds
  5. Taq polymerase binds to the double stranded side of DNA
  6. Temperature raised to 72 degrees, keeps the DNA single stranded. Taq catalyses the addition of nucleotides. starts at the primer then proceeds in the 5' to 3' end
  7. Amount of DNA increases exponentially
• Why is PCR time consuming

  You need to heat the DNA, let it denature and then it needs to cool to anneal
• Why is Taq polymerase more suitable than DNA polymerase in PCR

  • Obtained from thermophilic bacteria - more stable at higher temperature, optimum is 72
• Why are primers added

  DNA polymerase cannot bind to single stranded DNA
• Electrophoresis

  1. DNA samples digested using restriction enzymes at recognition sites at 30-40 degrees
  2. Tank is made using agarose gel and after its set buffer solution is added, with wells made at one end of the gel
  3. Loading dye is added to the tubes with digested DNA and then added to the wells
  4. Electrodes connected to a 18V battery and left to run for 6-8 hours. current to 5mA as there is a risk if electrical shock
  5. DNA fragments move at different speeds from the -ve to + +ve electrode - shorter fragments travel faster
• Issues relating to genetic manipulation

  .

  A) reduces

  B) Environment

  C) yield

  D) resistant

  E) common plant disease

  F) increase

  G) super weeds

  H) food waste

  I) reduces

  J) value

  K) demand

  L) conditions

  M) allergic

  N) Vitamin A, Zinc

  O) medicines

  P) vaccines

  
• DNA ligase

  catalyses the joining of sugar phosphate within DNA
• Electroporation

  introduce a vector w/ novel gene into a cell
• Plasmid

  loops of DNA in prokaryotic cells
• Recombinant DNA

  created in vitro by joining foreign DNA with a vector molecule
• Restriction Enzymes

  Endonuclease enzymes that cleave DNA molecules at specific recognition sites
• Vector

  carry/inserts DNA into another organism
• Gene therapy

  • introduces target gene into genome, which has now been transformed. target gene then transcribed and translated to produce desired protein
  • used to treat disease that are caused by mutation in a gene; if mutation is in a recessive allele, a wild-type dominate allele inserted. if mutation if in dominant then allele silences
• Somatic Gene Therapy

  Altering of alleles in adult body cells. short term and cannot be inherited
• Germline Cell Geen therapy

  Altering of alleles in sex cells. it is illegal in humans. long term and can be inherited
• Why might PCR produce less fragments than expected

  • primers fail to bind
  • temperature damages template strand
  • lack of free nucleotides
• DNA profiling

  relies on short, repeating sequences of DNA found in non-coding regions. VNTR; variable number tandem repeats. each invidiuval has a VNTR that differs in length resulting in unique DNA profile
• Why are introns used when profiling a human

  • Human genomes are similar
  • Coding regions of DNA wouldn't provide unique profiles
  • non-coding have VNTR - repeating sequences
• Genetic Engineering

  1. Isolate the gene - A; Restriction endonuclease, recognises specific recognition sites, creates sticky ends with exposed nucleotides. B; Reverse Transcriptase mRNA --> cDNA
  2. Insert into a vector; cut plasmid with the same RE. Produces the same sticky ends. DNA catalyses the phosphodiester bonds between plasmid and gene. Recombinant DNA.
  3. Transformation - making the bacteria plasma membrane more permeable. A; Ca2+ and heat shock. B; electroporation. C; electrofusion
  4. Mass production - agar gel with nutrients and ampicillin
• What percentage of the human genome is identical across all individuals?

  99.9 percent
• What causes variations between different individuals in the genome?

  The remaining 0.1 percent of the genome
• What do exons code for?

  Proteins
• What percentage of the genome do exons comprise?

  About 2 percent
• Why were introns previously called junk DNA?

  They were thought to have no function
• What is the current understanding of introns' role?

  They may regulate gene expression
• What is satellite DNA?

  Short repeated DNA sequences
• What is the length of mini-satellite DNA sequences?

  About 15 to 20 base pairs long
• How many times do mini-satellites repeat?

  50 to 100 times
• What is the length of microsatellite DNA sequences?

  About 2 to 4 bases long
• How many times do microsatellites repeat?

  5 to 15 times