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Microbiology 2230 3/22 and 3/24 lectures

by: Allison Collins

Microbiology 2230 3/22 and 3/24 lectures BIOL 2230

Marketplace > Middle Tennessee State University > Biology > BIOL 2230 > Microbiology 2230 3 22 and 3 24 lectures
Allison Collins
GPA 3.88

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These notes cover the notes from the week of 3/22. Topics covered include genetic probes, polymerase chain reactions, and recombinant DNA technology.
Anthony L Newsome
Class Notes
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This 5 page Class Notes was uploaded by Allison Collins on Thursday March 24, 2016. The Class Notes belongs to BIOL 2230 at Middle Tennessee State University taught by Anthony L Newsome in Fall 2015. Since its upload, it has received 61 views. For similar materials see Microbiology in Biology at Middle Tennessee State University.


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Date Created: 03/24/16
3/22   Genetic  Probe   •   Probes  can  be  made  to  identify  a  bacterium  or  a  genetic  disease   •   Ex:  Angelina  Jolie  got  a  double  mastectomy  and  hysterectomy  because  of   a  gene  sequence  found  by  a  genetic  probe  that  suggested  a   high   probability  of  breast  and  cervical  cancer   •   Probe:  a  DNA  or  RNA  molecule  which  is  used  to  locate  a  complementary   RNA  or  DNA  by  hybridizing  (through  complementary  base  pairing)  with  it   •   Hybrid  –  double  stranded  nucleic  acid  in  which  strands  differ  in  origin   •   ATCG  sequence  distinguishes  between  bacteria  (and  eukaryotes)   •   Manufacture  complementary  sequence  (as  a  guess)  and  if  it  binds  to   extracted  DNA  on  a  slide,  the  particular  ATCG  sequence  is  present     •   Indicator  molecule  (color)  –  color  change  on  slide  indicates  that  DNA   sequence  stuck  to  slide,  so  its  complementary  strand  is  present     DNA  synthesis  or  oligonucleotides  synthesis  (in  a  lab)   •   About  2-­‐30  bases  long   •   2  purposes   o   Probes  à  DNA  hybridization  à  identify  genes  or  bacteria   o   Primers  à  PCR  à  manufacture  copies  of  a  segment  of  DNA   In  situ  hybridization  –  using  genetic  probe  (oligonucleotides),  indicator   molecule   •   Cheaper  than  using  agar  plate;  common  process   •   Genetic  sequences  exist  that  are  associated  with  any  physical  condition     o   Vary  in  levels  of  directness  of  relationship  with  condition   Genetic  ID  card  –  23  &  Me   •   Cheap,  accessible   •   Indicates  the  increased  risk  of  diseases,  not  definite  occurrence   •   Drop  DNA  strand  onto  silicon  chip  and  test  for  attachment  to  genetic   probe   •   FDA  pulled  most  advertising  to  avoid  mass  panic/misunderstanding  by   the  public,  but  it  is  still  available  at  medical  centers   •   Ethical  questions   o   Do  insurance  companies  have  the  right  to  know  your  test   results?   o   Results  on  a  developing  fetus  could  affect  parents ’  decision  to   follow  through  with  pregnancy  or  more  testing   •   Can  also  associate  sequences  with  mental  issues  such  as  bipolar   disorder,  schizophrenia,  depression,  personality  traits   o   Does  an  employer  have  the  right  to  know?   •   Employers  have  been  found  to  secretly  test  blood  samples  in  some  cases   •   Horse  racing  –  “speed  gene”  test   Babies  –  triple  screen  test   •   High  percentage  of  false  positive  results   •   Genetic  counselors  can  help  analyze  results   •   FISH  –  fluorescent  in  situ  hybridization   o   Amniocentesis  –  extract  amniotic  fluid  –  risky  procedure   o   Double  check  triple  screen  for  accuracy   •   High  probability  that  you’ll  be  genetically  probed  at  some  point  in  your   lifetime   Polymerase  Chain  Reaction   •   Kary  Mullins  developed  it  and  won  Nobel  Prize  in  1993   •   Jurassic  Park  novel  based  on  PCR     2   3/24   Polymerase  Chain  Reaction   •   Relatively  simple  process   PCR  definition:  an  in  vitro  (in  a  test  tube)  reaction  in  which  a  specific  region   of  DNA  is  amplified  many  times  by  repeated  synthesis  of  DNA  using  DNA   polymerase  and  specific  primers  to  define  the  ends  of  the  amplified  region   •   Application:  make  large  quantities  of  a  particular  DNA  sequence   •   Primer  –  a  piece  of  DNA  that  provides  an  end  to  which  DNA  polymerase   can  add  nucleotides  –  approx.  nucleotides  long  in  this  process   Fundamental  steps   •   1.  Synthesize  fragments  with  sequences  (primers)  identical  to  those  on   either  side  of  the  targeted  sequence  (approx.  20  nucleotides)   •   2.  Denature  DNA  by  heating  it  to  94C  for  15  seconds   o   Separate  2  DNA  strands   •   3.  Add  multiple  primers  and  lower  temperature  to  68C  for  60  seconds  to   allow  primers  to  anneal  (hydrogen  bond)  to  DNA     o   Because  the  primers  are  added  in  excess,  the  the  targeted  DNA   strands  will  almost  always  anneal  to  the  primers  rather  than  to   each  other   •   4.  Add  nucleotide  triphosphates  and  DNA  polymerase   •   5.  The  DNA  polymerase  extends  the  primers  and  synthesizes  copies  of   target  DNA  sequence   o   2  strands  à  4  strands   •   6.  Repeat  heating  and  cooling  cycle  and  each  cycle  generates  a   complementary  strand  from  each  preexisting  strand   o   20  cycles  will  produce  about  1  million  copies   o   Pieces  ranging  in  size  from  <100  base  pairs  to  several  thousand   base  pairs  in  length  can  be  amplified   3   •   Use  a  heat  stable  polymerase  from  a  thermophilic  bacteria   –  only   polymerases  are  able  to  function  at  the  high  temps  used  in  the  PCR   o   Taq  polymerase  -­‐  from  Thermus  aquaticus,  bacteria  from  hot   springs     o   Vent  polymerase  –  from  Thermococcus  litoralis,  bacteria  from   deep  sea  vents   •   Forensics  application:  rape  case  in  Chattanooga   o   Semen  from  rape  victims  was  used  to  generate  RFLP  from  DNA   o   Cigarette  butt  from  rape  suspect  used  to  extract  small  amount   of  DNA  and  amplify  with  PCR     o   DNA  from  semen  matched  with  DNA  from  cigarette  butt,   suspect  convicted   o   Has  since  been  deemed  unlawful  to  confiscate  DNA  in  such  a   manner  unless  suspect  is  a  convicted  felon   Recombinant  DNA  technology  (rDNA)   •   Hepatitis  vaccine  –  originally  very  expensive,  today  is  free   o   Original  method:  extract  and  kill  virus  from  blood  sample  from   someone  who  is  Hepatitis  positive   o   Now:  take  nucleic  acid  from  virus,  introduce  to  bacteria,  bacteria   produces  proteins  à  use  protein  for  vaccine   rDNA  technology  in  bacteria  based  on:   •   1.  Restriction  endonucleases   o   found  in  bacteria  –  cut  DNA  (not  found  in  humans)   •   2.  DNA  ligases  (join  ends  of  DNA)   •   3.  Plasmids   •   4.  Gram  negative  bacteria   E.  coli  is  most  commonly  used  bacteria  in  biotechnology  (most  strands  are   nonpathenogenic)   4   •   Easy  to  grow   •   Dirt  cheap  to  grow   •   Grows  quickly   •   We  know  its  genetics  well  –  easy  to  place  genes  into  it  that  are   responsible  for  protein  production   Bacterial  production  of  proteins  used  for  vaccines   •   Take  cells  producing  desired  product  (eukaryotic  or  prokaryotic)   o   Isolate  DNA  and  treat  with  restriction  endonucleases   o   Some  of  the  DNA  fragments  have  desired  genes   •   Take  bacteria  resistant  to  ampicillin  (antibiotic)  and  resistance  carried  on   plasmids   o   Isolate  plasmids  and  break  open  with  restriction  enzymes     •   Mix  DNA  fragments  from  cells  with  desired  product  +  broken  plasmids   from  abx-­‐resistant  bacteria   •   Add  DNA  ligase  to  join  ends  together   o   Now  you  have  plasmids  with  antibiotic  genes  and  foreign  DNA   •   Screen  bacteria  for  production  of  desired  protein  and  product     o   Add  bacteria  (E.  coli)  sensistive  to  ampicillin  (i.e.  don’t  have   plasmids)  to  agar  plate  containing  ampicillin     §   Often  plasmids  don’t  get  into  bacteria  –  check  for   presence  on  an  agar  plate   §   Only  E.  coli  with  plasmids  (recombinants)  will  grow   §   Test  each  colony  on  the  agar  plate  for  proteins   §   One  colony  with  the  protein  means  success  –  can  now   duplicate  bacteria  from  that  colony         5  


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