reu 2014 poster presentation - jon deaton · poster by: jonathan deaton mentor: brian yu quake lab...

DNA  (bound  to  silicon)  

Silicon    membrane  

Centrifugal  Force  

DNA Extraction!

4.  Digital  PCR  (dPCR)  

Bacteria  frequently  harbor  mul7ple  copies  of  their  genome.  This  is  called  Polyploidy.  Griese  et  al.1  used  fluorescence  imaging  to  find  that  a  strain  of  cyanobacteria  called  Synechocys8s  PCC  6803  (Syn.  6803)  is  “highly  polyploid”,  with  over  200  copies  of  its  genome.  I  set  out  to  more  accurately  count  the  genome  copy  number  of  Syn.  6803  using  digital  PCR.  

2.  Designing  Primers  1.  Cell  Culture  &  DNA  Extrac8on  

My  research  this  summer  consisted  two  main  projects,  both  of  which  involved  the  characteriza7on  and  amplifica7on  of  microbial  genomic  DNA.    

Cyanobacteria  Shaker  Culture  

8  PCR  Primers  were  designed  for  100bp  amplicons,  evenly  distributed  throughout  the  genome  on  conserved  genes,  using  NCBI  genome  sequences  and  Primer  Blast®.  

Cyanobacteria  Syn.  6803  was  inoculated  into  test  tubes  and  grown  at  37°C  for  >7  days  before  call  harvest  and  extrac7on  of  genomic  DNA  using  lysozyme  and  silicon  membrane  centrifuga7on.  

Cita8ons  1.  Griese,  M.,  Lange,  C.  and  Soppa,  J.  (2011),  Ploidy  in  cyanobacteria.  FEMS  Microbiology  Le_ers,  323:  124–131.  doi:  10.1111/j.1574-­‐6968.2011.02368.x  

3.  Primer  Characteriza8on  Primer  efficiency  was  characterized  using  qPCR  and  linear  regression.  When100%  efficient,  DNA  quan7ty  (fluorescence)  will  double  ajer  each  PCR  temperature  cycle.  My  primers  were  characterized  to  be  approximately  100%  efficient.  

F  =  Fi  (2  –  ε)Cycle                    F  =  Fluorescence,  (2  –  ε)  =  Efficiency  

Digital  PCR  Ideology  

Digital  PCR  accurately  quan7fies  DNA  by  splilng  the  PCR  reac7on  into  many  (770)  chambers  such  that  only  some  chambers  contain  DNA  template.  During  thermocycling,  only  the  chambers  which  contain  DNA  template  will  undergo  PCR,  increasing  their  fluorescence.  DNA  is  quan7fied  by  coun7ng  these  chambers.  

•  Single  Syn.  6803  cells,  sorted  with  FACS,  were  lysed  with  lysozyme  at  37°C  •  Lysis  product  was  pre-­‐amplified  to  a  concentra7on  appropriate  for  dPCR  •  Pre-­‐amplifica7on  product  was  then  loaded  into  the  dPCR  microfluidic  chip  and  ran  •  Copy  number  was  calculated  using  dPCR  quan7fica7on,  accoun7ng  for  pre-­‐amplifica7on  

Results  and  Conclusions:      

Experiment  Design  

Ini7al  experiments  performed  using  all  8  primers  in  pre-­‐amp  and  dPCR  run  yielded  overabundant  DNA  concentra7ons,  a_ributed  to  amplifica7on  of  non-­‐genomic  DNA.  The  experiment  was  repeated  using  only  single  primers,  which  yielded  low  amounts  of  DNA  in  all  cases,  which  was  a_ributed  to  cells  not  lysing.  Lysis  dura7on  was  extended  to  18  hours  but  failed  to  yield  results.  Results  from  dPCR  using  extracted  gDNA  support  theory  of  failed  lysis.  

100bp  

330bp  

~100bp  Amplicons  

10bp

 DNA  Ladd

er  

50bp  

Electrophoresis  of  PCR  Product  of  Synechocys7s  PCC  6803  genomic  DNA      

ENGINEERING  Department  of  Bioengineering  

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Mul7ple  Displacement  Amplifica7on  (MDA)  is  an  isothermal  (30°C)  DNA  replica7on  method,  frequently  used  as  a  first  step  in  DNA  sequencing.  

Background  

•  May  amplify  genomic  DNA  of  unknown  sequence  •  Does  not  require  thermocycling  •  High  yield  and  genome  coverage  •  Low  error  frequency  

Advantages  

Disadvantages  •  Non-­‐specificity  may  lead  to  amplifica7on  of  DNA  contaminants  •  Amplifica7on  bias  is  generated  as  the  probability  of  amplicon  

amplifica7on  increases  with  amplicon  abundance.    •  Allelic  dropout,  the  non-­‐amplifica7on  of  one  of  a  pair  of  

heterozygous  alleles,  is  common      

Project  Goal  •  To  reduce  and  be_er  understand  

the  origin  of  amplifica7on  bias  

Approach  •  Perform  MDA  reac7on  in  a  microfluidic  

chamber  to  allow  for  the  mixing  of  reac7ons  with  different  biases  to  “even  out”  bias.  

Chip  Design  The  MDA  reac7on  mix  flows  into  the  

where  the  MDA  reac7on  takes  place.  The  reac7on  chamber  is  mixed  intermi_ently  using  the  

.  Ajer  ~3  hours  at  30°C  the  reac7on  product  may  be  retrieved  from  the    ports  by  pumping  TE  buffer  through  the  middle  ouplow  port.

Inflow  

Ouplow  

Ouplow

Ouplow

Ouplow  

Rotary  Pump  

Par77on  Control  

Par77o

n  Co

ntrol  

Par77on  Control  

Ouplow  

Results  and  discussion  My  work  consisted  of  fabrica7on,  tes7ng,  and  running  MDA  reac7ons  on  this  chip.  Previously,  issues  were  encountered  with  leaky  valves,  which  was  solved  by  the  dead-­‐end  filling  of  control  lines  with  water.  The  issue  of  “s7cky”  valves  (non-­‐opening)  was  addressed  by  reducing  control  layer  thickness  and  increasing  post  bonding  bake  7me.  While  this  reduced  “s7ckiness”,  it  allowed  for  the  control  membrane  to  tear  more  easily,  allowing  fluid  from  the  control  layer  to  leak  into  the  flow  layer.  Despite  these  issues,  three  MDA  reac7on  runs  were  performed  on  the  chips,  one  of  which  yielded  a  far  greater  quan7ty  of  DNA  product  than  the  others,  when  quan7fied  using  NanoDrop®.  The  quality  of  these  products  remains  to  be  further  determined  using  species  specific  PCR  primers.  However,  it  is  likely  that  contamina7on  of  some  kind  led  to  the  seemingly  large  quan7ty  of  MDA  product  in  one  of  the  reac7ons.  

Chip design by: Handuo Shi, Wen Torng, and Wanxin Wang!  

!

Reaction volume: ~ 0.1μL!

MDA  Reac?on  Ideology  

Poster by: Jonathan Deaton Mentor: Brian Yu Quake Lab jdeaton@stanford.edu

 This  research  was  made  possible  by  the  Stanford  Bioengineering  REU  Program  

Background