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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
|
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 [email protected]
This research was made possible by the Stanford Bioengineering REU Program
Background