chapter 21

• DNA sequencing first methods

  involved radioactively labelled bases and gel electrophoresis, all done by hand so very time consuming and the sequence was read manually from bottom of gel to the top, this was Sanger sequencing

  
• sanger sequencing

  the radioactive labels were then changed for fluorescent tags which could be read automatically by a machine, a laser detected each tag on the DNA base as it ran through the gel past a sensor in the machine

  
• later versions of DNA sequencing used a gel in a capillary tube to run the sample through, many samples could be prepared and sequenced in one go, order of bases are then read by a computer

  
• genome

  all of the genetic material it contains
• exons

  2% of total DNA that code for proteins
• introns

  large non-coding regions of DNA that are removed from mRNA before it is translated into a polypeptide chain
• satellite DNA

  short sequences of DNA that are repeated multiple times within introns, telomeres and centromeres
• minisatellite DNA

  sequence of 20-50 base pairs that are repeated 50 to 100 times, occur at more than 1000 locations in the human genome and are known as variable number tandem repeats (VNTRS)
• microsatellite DNA

  smaller region than a minisatellite of just 2-4 bases repeated only 5-15 times, also known as short tandem repeats (STRS), always appear on the same positions on the chromosomes but the number of mini or microsatellites vary between individuals so different lengths of repeats are inherited from both parents
• identical twins will have identical coding and an identical satellite pattern, but also the more closely related you are to someone the more likely you both share similar patterns
• patterns in non coding DNA were discovered by Professor Sir Alec Jeffreys at Leicester Uni in 1984, they produced an image of patterns in the DNA of an individual called DNA profiling, technique used by scientists to assist in the identification of individuals or familiar relationships
• producing a DNA profile

  process contains 5 steps:

  1. extracting the DNA
  2. digesting the sample
  3. separating the DNA fragments
  4. hybridisation
  5. seeing the evidence
• DNA profiling 1. extracting the DNA

  DNA must be extracted from a tissue sample using a technique called the polymerase chain reaction (PCR) and a small fragment of tissue will give a large enough sample

  
• DNA profiling 2. digesting the sample

  strands of DNA are cut into small fragments using the special enzymes called restriction endonucleases, different restriction endonucleases cut DNA at a specific nucleotide sequence known as a restriction site, all restriction endonucleases make 2 cuts, once through each strand of the double helix, there are many different restriction endonucleases

  
• DNA profiling 3. separating the DNA fragments

  to produce a DNA profile, the cut fragments of DNA need to be separated to form a clear and recognisable pattern, done by using electrophoresis, charged particles move through a gel medium under the influence of an electrical current, gel immersed in alkali in order to separate DNA double strands and the single stranded DNA fragments then transferred onto a membrane