Luke  Droney  

Molecular  Techniques  Explain  in  general  terms  various  molecular  techniques  

1)   DNA  and  RNA  extraction  2)   Polymerase  chain  reaction  3)   Hybridisation  assays  4)   Sequencing  

     DNA  and  RNA  extraction    

•   Specimen  processing  involves  liberating  the  nucleic  acid  from  the  target  of  interest  and  the  removal/inactivation  of  potential  inhibitors  of  nucleic  acid  amplification/hybridization  

•   Amount  of  manipulation  required  is  dependent  upon  specimen  type,  sensitivity  required  and  amount  of  specimen  available.  

o   CSF,  blood,  serum  or  plasma  are  relatively  uncomplicated  to  process  

o   Tissue,  bone,  sputum  or  stool  more  difficult  o   Mycobacteria  and  fungi  are  difficult  to  process  compared  to  

viruses.  •   In  a  specimen  of  known  nucleic  acid  content  and  without  strong  

inhibitors  of  amplification  the  process  may  be  as  simple  as  heating  to  100  degrees,  spinning  to  remove  insoluble  material,  then  proceeding  with  the  assay  on  the  supernatant.  

•   Semi-­‐‑automated  and  automated  instruments  have  significantly  simplified  processing  procedures  for  more  complex  specimens.  

 DNA  purification;  

1.   Effective  disruption  of  cells  or  tissue;  2.   Removal  of  membrane  lipids  with  detergent  3.   Denaturation  of  nucleoprotein  complexes  (using  a  protease);    4.   Removal  of  RNA  with  an  RNAse  5.   Purify  DNA  by:  

a.   Ethanol  –  DNA  is  alcohol  insoluble  and  will  form  a  pellet  on  centrifugation.  

b.   Phenol-­‐‑chloroform  extraction  c.   Microcolumn  purification  –  DNA  binds  to  solid  phase  dependent  

up  pH/salt  content  of  the  buffer.    RNA  extracted  by  Guanidinium  Thiocyanate-­‐‑Phenol-­‐‑Chloroform  Extraction.    Hybridisation  assays    The  use  of  DNA  probes  to  directly  detect  or  characterize  a  target  is  well  standardized  and  simpler  than  other  techniques  but  lacks  sensitivity  in  comparison  to  enzyme  amplification/sequencing  technologies.    Solid-­‐‑phase  hybridization:  

Luke  Droney  

•   Intact  cells  are  lysed  and  DNA  is  denatured  •   DNA  is  brought  into  contact  with  and  fixed  to  a  nylon  membrane  •   Membrane  is  immersed  in  hybridization  solution  containing  the  DNA  

reporter  probe  and  allowed  to  hybridize.  •   Incubate  (slow  and  usually  requires  overnight  incubation)  •   Unbound  reporter  probes  are  washed  away  and  bound  probe  is  detected.  •   Some  assays  use  a  sandwich  approach  where  a  capture  probe  is  bound  to  

the  membrane  and  the  nucleic  acid  detected  by  a  second  labeled  probe.  •   E.g  Southern  blot  (DNA  –  e.g  TCR  gene  rearrangement  studies)  or  Northen  

blot  (RNA)    Solution-­‐‑phase  hybridization:  

•   Target  nucleic  acid  and  probe  interact  in  an  aqueous  reaction  mixture.  •   Rapid  hybridization  kinetics  –  less  than  1  hour  to  complete.  •   Nucleic  probe  must  be  single  stranded  and  not  hybridize  with  itself.  •   E.g  hybridization  protection  assay  –  (HPA  –  used  to  detect  bacterial  or  

fungal  nucleic  acid).  Hybridization  of  the  probe  (with  a  chemiluminscent  label)  and  target  prevents  breakdown  of  the  probe  by  addition  of  alkali  solution.  Peroxides  are  added  and  chemiluminescence  is  measured.  

 In-­‐‑situ  hybridization:  

•   Similar  principle  to  solid  and  solution-­‐‑phase  hybridizations.  •   However,  occurs  within  the  context  of  intact  morphology  to  report  the  

presence  of  the  target  within  tissues.  •   Sensitivity  limited  by  ability  to  target  intracellular  structures  (smaller  

probes  are  optimal  to  favour  tissue  penetration)      Nucleic  acid  amplification  techniques    PCR:  

•   Utilises  the  ability  of  DNA  polymerase  to  copy  a  strand  of  DNA  by  elongation  of  complementary  strands  initiated  by  a  pair  of  closely  spaced  oligonucleotide  primers.  

•   Each  cycle  of  the  reaction  will  double  the  amount  of  target  DNA,  resulting  in  exponential  levels  of  DNA  amplification  (in  theory  as  few  as  20  cycles  could  yield  approximately  1  million  times  the  amount  of  target  DNA  initially  present).  

 (Below  diagram  from  Molecular  and  clinical  Laboratory  Immunology  pg  31)    (variations  on  this  technique  use  a  reverse  transcriptase  step  to  detect  RNA)    

Luke  Droney  

   

       

Luke  Droney  

Post-­‐‑amplification  detection:  •   Detection  of  PCR  products  requires  less  stringent  hybridization  than  

detection  of  unamplified  nucleic  acid  due  to  large  amounts  of  homogenous  product.  

•   Amplified  DNA  is  captured  by  probes  attached  to  a  microtitre  plate  or  magnetic  beads.  

•   Incorporation  of  biotin  or  other  detection  system  into  the  amplified  DNA  allows  detection  with  labeled  probe  (e.g  avidin-­‐‑HRP).  

 Real-­‐‑time  product  detection:  

•   Fluorescence  emission  of  a  reporter  probe  is  measured  cycle  by  cycle  (increased  by  accumulation  of  PCR  product)  

•   Utilises  one  instrument  (thermal  cycler/signal  detector)  for  both  amplification  and  signal  detection.  

•   Closed  system  –  (i.e  no  transfer  of  products  to  another  plate/reader)  –  less  chance  of  contamination.  

 Taqman:  

•   Utilises  a  short  sequence  nucleotide  reporter  probe  specific  for  target  sequence  with  a  5’  fluorescent  molecule  and  a  3’  quencher  molecule  (i.e  under  normal  circumstances  fluorescence  is  prevented  by  the  close  proximity  of  the  quencher).  

•   When  DNA  extension  from  the  primer  occurs,  the  Taq  exonuclease  cleaves  the  5’  end,  releasing  the  fluorescent  molecule.  Fluorescence  is  proportional  to  the  amount  of  PCR  product.  

•   Multiple  targets  in  the  same  solution  can  be  detected  through  the  use  of  different  fluorescent  dyes.  

 (diagram  from  pg  37  Molecular  and  clinical  Laboratory  Immunology)    

Luke  Droney  

     Hybridisation  probes:  

•   Two  free  probes  are  used  which  bind  to  adjacent  sections  of  amplified  product.  

•   The  5’  probe  has  a  bound  fluorescein  molecule  and  a  3’  acceptor  probe  has  a  red  reporter  dye.  

•   During  the  annealing  step  the  probes  hybridize.  With  excitation  of  the  fluorescein  dye,  the  excitation  is  transferred  to  the  red  reporter  and  red  fluorescence  is  emitted.  

 

Luke  Droney  

     Sequencing    Sanger  sequencing  (‘First  generation’  sequencing):  

•   Most  accurate  currently  available  sequencing  technology  •   Uses  fluorescently  labeled  2’,3’-­‐‑dideoxynucleoside  triphosphates  (A,  C,  G,  

T  labeled  with  a  different  fluorescent  dye)  •   Dideoxynucleoside  triphosphates  are  similar  to  normal  nucleosides  but  

terminate  DNA  polymerase  transcription  at  the  point  at  which  they  are  incorporated.  

•   A  reaction  is  carried  out  in  a  single  tube  containing  the  DNA  to  be  sequenced,  normal  nucleosides  (2’-­‐‑deoxynucleoside  triphosphates)  and  fluorescently  labeled  dideoxynucleoside  triphosphates.  

•   When  the  reaction  is  finished  the  tube  contains  multiple  nucleotide  fragments  of  variable  length,  each  having  a  common  5’  end  and  a  variable  3’  end  (depending  on  which  fluorescently  labeled  dideoxynucleotide  triphosphate  is  3’).  

•   After  removal  of  excess  free  nucleotides  the  fragments  are  run  in  a  gel  or  capillary  –  the  smallest  fragments  are  fastest  moving.  Detection  of  the  fluorescent  labels  allows  determination  of  the  sequence.  

•   Sanger  sequencing  requires  a  single  strand  DNA  template.  •   The  biggest  limitation  of  Sanger  sequencing  is  throughput,  particularly  

due  to  the  electrophoresis  stage.  

Luke  Droney  

   Next-­‐‑generation  sequencing:  

•   Refers  collectively  to  the  new  high-­‐‑throughput  techniques  that  allow  large  numbers  of  sequencing  reactions  to  occur  simultaneously.  

•   Next  generation  techniques  monitor  the  addition  of  nucleotides  to  immobilized/spatially  arrayed  DNA  templates.  

 Steps  in  next-­‐‑gen  sequencing  (pictures  from  http://www.illumina.com/content/dam/illumina-­‐‑marketing/documents/products/illumina_sequencing_introduction.pdf):  

1.   Fragmentation/size  selection  –  DNA  is  randomly  broken  down  into  smaller  sequence-­‐‑able  fragments.  

2.   5’  and  3’  adapter  ligation  adds  platform-­‐‑specific  synthetic  DNAs  to  the  ends  of  fragments,  which  serve  as  primers  for  downstream  amplification  and/or  sequencing  reactions.  

 3.   Templates  are  immobilized/spatially  separated  and  amplified.  

a.   Errors  can  be  introduced  in  this  stage  due  to  the  imperfect  nature  of  DNA  polymerase,  which  can  create  problems  at  the  data  analysis  stage.  

Luke  Droney  

b.   Some  platforms  can  sequence  without  amplification  4.   Cluster  generation  –  amplification  products  are  captured  on  a  solid  

surface  of  oligonucleotides  complementary  to  the  adapters.  5.   Each  fragment  is  amplified  into  clonal  clusters  by  bridge  amplification.  

 6.   Sequencing;  

a.   Fluorescently  labeled  nucleotides  are  added  at  each  cluster  and  ‘read’  with  each  addition.  

Luke  Droney  

 7.   Alignment  and  data  analysis;  

a.   Fragment  reads  are  aligned  with  a  reference  genome.    

Luke  Droney  

       I  found  this  Illumina  video  useful  in  understanding  the  process.  Keep  in  mind  that  different  platforms  use  different  amplification/sequencing  techniques:  https://www.youtube.com/watch?v=HMyCqWhwB8E&list=UUxWMU29FF4kIG8YmQf6Zv0g      Coverage  and  error  rates;  

•   Because  of  error  rates  in  DNA  polymerase,  it  is  important  that  there  is  sufficient  ‘coverage’  of  the  genomic  area  of  interest  to  ensure  SNPs  are  correctly  identified.    

 

Luke  Droney