Topic 7

• Hersey and Chase


  To test if genetic material is is DNA or proteins: viruses were grown in two isotopic mediums in order to label a specific component as radioactiveViruses grown in radioactive sulphur had radiolabelled proteins (sulphur is present in proteins but not DNA)Viruses grown in radioactive phosphorus had radiolabeled DNA (phosphorus is present in DNA but not proteins)The viruses were then allowed to infect a bacterium and then the virus and bacteria were separated via centrifugationThe bacterial pellet was found to be radioactive when infected by the P-viruses (DNA) but not the S-viruses (protein)This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria

  
• Rosalind Franklin's and Maurice Wilkins' investigation of DNA structure by X-ray diffraction


  - DNA was purified and then fibres were stretched in a thin glass tube (to make most of the strands parallel)- The DNA was targeted by a X-ray beam, which was diffracted when it contacted an atom- The scattering pattern of the X-ray was recorded on a film and used to elucidate details of molecular structure
• X ray diffraction suggested


  Bi-directional replication (anti-parallel strands) and Complementary base pairs
• Pyramidines


  thymine and cytosine
• Purines


  Adenine and Guanine
• Purines make up...


  60%
• Pyramidines make up...


  40%
• Why Prunines A and G?


  A and G = silver = pure
• DNA replication is a...


  semi-conservative process that is carried out by a complex system of enzymes
• Helicase


  An enzyme that unwind and seperates the double helix at the replication forks, by breaking the hydrogen bonds between bases, this occurs at the origin of replication forming a replication fork
• DNA Gyrase


  Enzyme that temporarily breaks the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix
• single stranded binding proteins


  keep the strands of DNA separate during replication, stops from reannealing
• DNA primase


  synthesis of RNA primer
• DNA polymerase III


  In charge of aligning free nucleotides with the complementary base pair, attaches at 3' end, synthesises in a 5' to 3' direction
• DNA polymerase I


  removes the RNA primers from the lagging strand and replaces them with DNA nucleotides
• DNA ligase


  an enzyme that eventually joins the sugar-phosphate backbones of the Okazaki fragments
• leading strand


  The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.
• lagging strand


  A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork.
• DNA polymerase adds nucleotides at the


  3' end
• cleaving of 2 phosphates =


  hydrolysis reaction
• DNA is synthesized in what direction?


  5' to 3
• DNA replication

  
• DNA polymerase III can only add nucleotides to what end?


  3
• On the leading strand, DNA polymerase is moving towards...


  the replication fork and so can copy continuously
• On the lagging strand, DNA polymerase is moving away from...


  the replication fork, meaning copying is discontinuous
• Okazaki fragments


  Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.

  
• DNA sequencing


  the process of determining the precise order of nucleotides within a DNA molecule
• dideoxyribonucleic acid is used for...


  preparing samples for base sequencing
• Dideoxynucleotides


  Lack an 'OH, chain terminator, no more elongation (v. deoxy which has 'OH group that can extend)
• dideoxyribonucleic acid


  stops DNA replication for base sequencing
• Method of DNA sequencing


  Sanger Method
• Sanger sequencing step by step (summarised)


  - Dideoxynucleotides can be used to determine DNA sequence using the Sanger method1. 4 dideoxynucleotides for each of the bases are made and is combined with normal bases - whenever the didi is incorporated the DNA sequence is terminated at that base position e.g. when the ddA is mixed it will stop at all the A bases2. 1 complete PCR cycle generates millions of sequences, so every base position is likely to be terminated 3. these sequences are then separated via gel electrophoresis into their bases4. Automated machines determine the sequence quickly when dideoxynucleotides have been made radioactive or florescent - the opposite strand is now know, (If the Sanger method is conducted on the coding strand (non-template strand), the resulting sequence elucidated will be identical to the template strand)
• Sanger Sequncing method with didgi nucleotides

  
• gel electrophoresis


  Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel
• PCR


  (polymerase chain reaction) multiple copies of a specific segment of DNA
• Non-coding DNA


  a region of DNA that does not contain a sequence of nucleotides that will be expressed, but are still useful e.g. telomeres, introns, non-coding RNA, tandem repeats - only 1.5% of DNA is involved in coding for proteins
• Intron


  sequence of DNA that is not involved in coding for a protein (intruding)
• Exon


  expressed sequence of DNA; codes for a protein (expressing)
• Short Tandem repeat


  Within the non-coding regions of an individual's genome there exists satellite DNA - long stretches of DNA made up of repeating elements called short tandem repeats (STRs)
• Short Tandem Repeats are used in


  DNA profiling