qpcr: Rapid Method Tour

Transcription

1 qpcr: Rapid Method Tour Save The Harbor/Save The Bay May 30,

2 Overview PCR - Problem PCR - Background & How it Works PCR - Correlations PCR - Present & Future 2

3 Mommy? When will we know if it s safe to swim in the water? Problem: Bacterial culture methods yield Results NEXT day. Solution : Reliable rapid methods inform bathers of Water Quality the SAME day. Sweetheart, we won t really know until tomorrow. 3

4 Background Polymerase Chain Reaction (PCR) is an analytical tool applied from the branch of science called Genomics. Genomics is the study of organisms in terms of their DNA sequences and cellular activity in response to the environment. 4

5 Background 5

6 Background -The Inventor Dr. Kary Mullis received a Nobel Prize in chemistry in 1993 for his invention of the polymerase chain reaction (PCR). Conceptualized in 1983 by Dr. Mullis, The process is hailed as one of the monumental scientific techniques of the 20th century. 6

7 How It Works Genomics is the science of gene Identification Structure, and Function 7

8 PCR is AKA: Repetitive Molecular Photocopying Utilizes Polymerase: natural enzyme catalyst DNA replication Analytical method that mimics the process of cellular DNA duplication Method Premise - Many copies are easier to identify than fewer copies TV Crime Shows: NCIS, CSI-Miami, etc. 8

9 How It Works Polymerase Chain Reaction (PCR): - uses the natural functions of the enzyme polymerase - to copy, genetic material (e.g., DNA or RNA) - in a simple 3 step process: 1. Denaturation 2. Annealing 3. Extension The goal of PCR is to generate millions - billions of copies of target gene sequences through repetitive cycling of the 3 PCR steps 9

10 WHY AMPLIFY DNA? In traditional microbiology bacterial cells are grown or amplified to form colonies which can be easily identified and counted. In PCR sections of DNA, unique target gene sequences, are copied or amplified instead of whole cells. Target 10

11 How It Works 4th cycle Target Gene 3rd cycle PCR Cycles minutes 2nd cycle From 1 original copy 1st cycle 2 n copies per cycle To Millions or Billions of Copies 2 35 = 34 Billion Copies 11

12 Detection & Concentration 12

13 A Word About Correlations Be Careful Know Why qpcr & cult. results corr. are Site specific Temporally specific qpcr & epidemiology corr. are More important Show higher corr. than 13

14 Enterococci Concentration 10,000,000 1,000,000 Enterococcus / 100mL 100,000 10,000 1, Site PCR CCE PCR CFU Enterolert MPN 14

15 qpcr & Epi Data 15

16 2012 Published new EPA qpcr Recreational Water Method with DNA-based Rec Water Criteria Source Tracking Apps & Research cont d 2012 egst RARE Human Vs. Nonhuman Fecal Indicator Organisms (FIOs) Tool Box 2013 qpcr Method Training 16

17 References & Links EPA Genomics Task Force White Paper Inside PCR R%20Virtual%20Lab Kary Mullis: 17

18 Questions 18

19 PCR Sample to Results 19

20 How We Use It PCR is a powerful analytical tool for supporting a broad spectrum of EPA Programs: Superfund: Risk Assessment (e.g., Estrogen, Hg, etc.) Bioremediation NPDES: Toxicity Testing TSCA: Nanotechnology TMDL: Microbial Source Tracking Source Identification CAFOs Drinking Water: Rapid Screening BEACHES: Rapid Water Testing 20

21 Robustness 21

22 Specificity 22