bio382-f14 lab #3 pcr new - mdcune · bio382-f14 lab #3 pcr new.pptx author: keller, lani c. prof....

Polymerase  Chain  Reac1on  

Bio382-­‐F14  Lab  #3  

Dr.  Lani  Keller    

Cool  PCR  Learning  Tools:  hCp://learn.gene1cs.utah.edu/content/labs/pcr/  

hCp://www.dnalc.org/resources/anima1ons/pcr.html        

Enzyma1c  amplifica1on  of  a  specific  DNA  fragment,  using  repeated  cycles  of  denatura1on,  primer  annealing,  and  chain  extension.    

Polymerase  Chain  Reac1on  (PCR)  

hCp://www.karymullis.com/  

•  DNA  cloning  •  DNA  sequencing  •  Disease  gene  tes1ng  •  Forensics  •  Paternity  tes1ng  •  Gene  mutagenesis    •  Evolu1onary  gene1cs  

PCR  Replica1on  Cycles  

Several  Repe**ve  Steps:    1.  94-­‐98°C    denaturing  “mel1ng”  DNA  into  two  single  

strands.      2.  50-­‐65°C    annealing  of  primers  to  complementary  

sequences  (by  hydrogen  binding)  on  either  side  of  the  target  sequence.  Variable  temperature.  

 3.  72°C    extension  by  DNA  polymerase  binding  and  

synthesizing  a  cDNA  strand  from  each  primer.    

Figure  4.1  Polymerase  chain  reac1on:  first  cycle.  Figure  4.2  Polymerase  chain  reac1on:  second  cycle.  

1.      A  thermo-­‐stable  DNA  polymerase  (Taq  polymerase)  Prior  to  this  discovery,  you  would  have  to  add  new  DNA  polymerase  each  cycle  because  it  would  become  inac1ve  during  the  high-­‐temp  denatura1on  step.    

Two  Important  Innova1ons  

2.   Thermo-­‐cyclers  Before  computers  controlled  the  repe11ve  temperature  changes,  people  would  literally  set  up  three  water  baths  and  manually  move  the  tubes.    

1.  Reac1on  buffer    Provides  suitable  chemical  environment.    

2.  Deoxynucleo1de  triphosphates  (dNTPs)    Building  blocks  for  DNA  synthesis.  Nucleo1des  with  triphosphates.    

3.  DNA  template    A  DNA  source  containing  target  DNA  to  be  amplified.      

4.  Forward  and  Reverse  primers  or  “oligos”    Small  pieces  of  DNA  that  are  complimentary  to  the  target  DNA    

5.  DNA  polymerase    A  thermostable  DNA  polymerase  (op1mum  temperature  ~70°C)  

Five  Main  Components  of  PCR  

The  Template  DNA  

3.  Genomic  RNA  à  cDNA  •  Isolate  RNA,  make  cDNA  by  reverse  transcrip1on  

•  Can  look  at  what  genes  are  being  expressed    

2.    Genomic  DNA  •  RNA  contamina1on  causes  problems  •  Contains  exons  and  introns    •  A  lot  of  non-­‐target  DNA  •  Need  1ng  –  1µg  as  template  

1.    Recombinant  plasmid  DNA  •  Most  efficient  •  Need  1pg  –  1ng  as  template  

Primer  Design  

Figure  4.3  Forward  and  reverse  primers.  

•  18-­‐28bp  of  homology  specific  to  target  DNA  •  Op1mal  annealing  temps  (~58°C)  should  be  similar  for  both  

forward  and  reverse  primers  

1.  Whole  genome  à  NO  2.  En1re  genes  à  Not  usually  3.  Pieces  of  genes  with  SNPs  à  YES!  4.  Can  you  have  errors?  à  YES!  5.  If  you  add  100-­‐1mes  the  amount  of  star1ng  

template  DNA,  do  you  expect  to  have  100-­‐1mes  the  amplified  DNA  à  NO    

6.  We  amplified  piece  of  mtDNA  and  will  cut  it  with  an  endonuclease  to  examine  your  haplotype!  

What  do  you  actually  amplify?  

hCp://www.ncbi.nlm.nih.gov/nuccore/251831106    

Order  of  lab  today  1.  Start  with  procedure  D  (restric1on  digest  of  PCR  

products).  We  will  start  diges1ons  as  soon  as  possible.    

2.  During  1  hour  incuba1on:  start  on-­‐line  PCR  module  

3.  Amer  1  hour  incuba1on:  prepare  and  load  gels.  Each  person  will  run  uncut  PCR  product  (5  uL  PCR/loading  dye  mixture)  AND  digested  PCR  product  (30  uL  digest  +  5  uL  loading  dye    )  

4.  While  loading-­‐  finish  the  PCR  module,  turn  in  Excel  file,  and  take  on-­‐line  post  test  

Addi1onal  PCR  Resources  and  Anima1ons  for  Students  

•  hCp://www.dnalc.org/resources/anima1ons/pcr.html  •  hCp://learn.gene1cs.utah.edu/content/labs/pcr/  •  hCp://www.sumanasinc.com/webcontent/anima1ons/content/pcr.html  •  hCp://highered.mheduca1on.com/sites/0072556781/student_view0/

chapter14/anima1on_quiz_6.html  •  hCp://www.lifetechnologies.com/us/en/home/life-­‐science/pcr/elevate-­‐

pcr-­‐research/pcr-­‐video-­‐library/pcr-­‐anima1on.html  •  hCp://highered.mheduca1on.com/olc/dl/120078/micro15.swf  •  hCp://www.promega.com/resources/mul1media/pcr/introduc1on-­‐to-­‐pcr/