Release date: November 2015 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Food Safety Lab Science for everyone,

Slides:



Advertisements
Similar presentations
Welcome! Our goals for today
Advertisements

Polymerase Chain Reaction (PCR) and its Applications.
PCR way of copying specific DNA fragments from small sample DNA material "molecular photocopying" It’s fast, inexpensive and simple Polymerase Chain Reaction.
COMPUTER EXERCISE Design of PCR and PCR-RFLP experiments This presentation shows all steps of a PCR-RFLP experiment and is a companion of the computer.
DNA Fingerprinting and Forensic Analysis Chapter 8.
Definition of PCR Requirements for PCR PCR Process Agarose gel electrophoresis.
Introduction to DNA.
Release v3.2: February 2015 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Crime Lab Science for everyone,
General Genetics. PCR 1.Introduce the students to the preparation of the PCR reaction. PCR 2.Examine the PCR products on agarose gel electrophoresis.
Genomic DNA purification
Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler June 2009 Chapter 7 DNA Amplification.
Bioinformatics/PCR Lab How does having a certain genetic marker affect chances of getting brain cancer?
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) and its Applications.
CULTURE INDEPENDENT ANALYSIS OF MICROBIAL COMMUNITIES IN SOIL
Polymerase Chain Reaction (PCR)
Recombinant DNA Technology………..
Genetics Techniques: RFLP & PCR AP Biology Unit 3.
Basic methods in genetics PCR; Polymerase Chain Reaction Restriction enzyme digestions Gel electrophoresis.
Module 1 Section 1.3 DNA Technology
The Polymerase Chain Reaction
PCR Forensics. Today’s Lab There has been an outbreak of Salmonella poisoning in the Student Union cafeteria at Stanford University cafeteria. You have.
Polymerase Chain Reaction (PCR) Developed in 1983 by Kary Mullis Major breakthrough in Molecular Biology Allows for the amplification of specific DNA fragments.
Got Milk? SNPs, Inheritance, and the Evolution of Lactose Tolerance.
Release v2.4: January 2015 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Crime Lab Science for everyone, everywhere.
Molecular Testing and Clinical Diagnosis
Polymerase Chain Reaction (PCR)
The polymerase chain reaction
Bioinformatics & Biotechnology Lecture 1 Sequencing BLAST PCR Gel Electrophoresis.
Human Genomics. Writing in RED indicates the SQA outcomes. Writing in BLACK explains these outcomes in depth.
The polymerase chain reaction
Release v3.3.2: November 2015 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Crime Lab Science for everyone,
Polymerase Chain Reaction A process used to artificially multiply a chosen piece of genetic material. May also be known as DNA amplification. One strand.
Amplification of a DNA fragment by Polymerase Chain Reaction (PCR) Ms. Nadia Amara.
Molecular Genetic Technologies Gel Electrophoresis PCR Restriction & ligation Enzymes Recombinant plasmids and transformation DNA microarrays DNA profiling.
PCR With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies.
Crime Scene Investigator PCR Basics™
The Polymerase Chain Reaction (PCR)
Introduction to PCR Polymerase Chain Reaction
Bringing DNA science to more places
Release v3.3.3: March 2016 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Crime Lab Science for everyone, everywhere.
Polymerase Chain Reaction (PCR). What’s the point of PCR? PCR, or the polymerase chain reaction, makes copies of a specific piece of DNA PCR allows you.
Release v3.3.1: March 2016 © Copyright by Amplyus LLC, all rights reserved miniPCR TM Food Safety Lab Tainted Patties! Science for everyone, everywhere.
The Search for a Jumping Gene: Module based on a kit from Bio-Rad Laboratories, Inc. Adapted by Dan Murray from a presentation by: Stan Hitomi - Monte.
1/03VisTE Project - Copyright 2002 Biotechnology The Polymerase Chain Reaction (PCR)
Lecturer: Bahiya Osrah Background PCR (Polymerase Chain Reaction) is a molecular biological technique that is used to amplify specific.
Rajan sharma.  Polymerase chain reaction Is a in vitro method of enzymatic synthesis of specific DNA sequences.  This method was first time developed.
PCR Polymerase chain reaction. PCR is a method of amplifying (=copy) a target sequence of DNA.
Polymerase Chain Reaction. Before PCR Before PCR Recombinant Recombinant DNA DNA technology technology.
DNA amplification and analysis: miniPCR TM Crime Lab Release v4.0: October 2016 © Copyright by Amplyus LLC, all rights reserved.
Introduction to PCR Polymerase Chain Reaction
Polymerase Chain Reaction
Lab 8: PCR (Polymerase Chain Reaction)
PCR TECHNIQUE
COURSE OF MICROBIOLOGY
Polymerase Chain Reaction
Polymerase Chain Reaction
PCR and RLFP’s.
DNA profiling DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles. Definitions you will.
DNA amplification and analysis: miniPCRTM GMO Lab Heart-Shaped Bananas
BIOTECHNOLOGY BIOTECHNOLOGY: Use of living systems and organisms to develop or make useful products GENETIC ENGINEERING: Process of manipulating genes.
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) technique
Sequencing and Copying DNA
Recombinant DNA Unit 12 Lesson 2.
Introduction to Polymerase Chain Reaction (PCR)
PCR Polymerase chain reaction (PCR)
PCR DNA fingerprinting Gel electrophoresis
SBI4U0 Biotechnology.
Principles of Quantitative PCR
Presentation transcript:

Release date: November 2015 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Food Safety Lab Science for everyone, everywhere

1 © Copyright by Amplyus LLC, all rights reserved Welcome Our goals for today: Review DNA amplification theory Solve a public health problem using DNA analysis

2 © Copyright by Amplyus LLC, all rights reserved Polymerase Chain Reaction (PCR) A process that identifies and copies (amplifies) a specific piece of DNA in a biological sample Complex DNA sample Amplified DNA (Billions of copies) Region of interest  Sequencing  Genetic risk  Pathogen detection  Drug development  Crop modification  Forensic analysis  Etc. Applications

3 © Copyright by Amplyus LLC, all rights reserved PCR relies on DNA’s unique structure Source: US National Library of Medicine, NIH, Thinkquest DNA: a double helix......held together by base complementarity

4 © Copyright by Amplyus LLC, all rights reserved How PCR works: 3 steps to copy DNA Denaturation 1 94°C Annealing °C Primer 2 Primer 1 Extension 3 72°C Taq DNA polymerase dNTPs

5 © Copyright by Amplyus LLC, all rights reserved How PCR works: repeat the cycle DenaturationAnnealingExtension DNA + primersdenatured DNADNA + copy 94° C 50-60° C 72° C Repeat x ~25-30 cycles Single molecule~1B copies

6 © Copyright by Amplyus LLC, all rights reserved PCR makes DNA visible (and useful)

7 © Copyright by Amplyus LLC, all rights reserved PCR Text PCR is central to biomedical applications Forensics Food and agriculture Consumer genomics Molecular diagnostics Personalized medicine Human evolution Text

8 © Copyright by Amplyus LLC, all rights reserved Outbreak of E. coli in ground beef  Dozens of victims nationwide Ongoing USDA investigation  Search for culprit meat processing plant  Test DNA samples for E. coli O157:H7 Use of core biotechnology techniques  PCR  Restriction digest  DNA electrophoresis Demonstration of real-world impact  DNA in public health / surveillance  Biotechnology in food industry We will use DNA technology to solve a real-world problem

9 © Copyright by Amplyus LLC, all rights reserved Scientists work hard at detecting foodborne pathogens Most Escherichia coli are harmless and live in our gut Pathogenic strains can be serotyped by O/H antigens 0157:H7 strains can cause severe hemorrhagic diarrhea Complications can cause kidney damage and eventually, death Bacterial culture and antigen detection can be slow and fairly insensitive

10 © Copyright by Amplyus LLC, all rights reserved Outbreaks are a serious public health concern Outbreaks of E. coli O157:H7 occur Every year A single outbreak can last Several months O157:H7 infection results in 2,100 hospitalizations annually A single outbreak can lead to recalls of millions of pounds of food PCR analysis can serve to detect and stop the spread of infection Why is PCR useful? PCR is ideal for fast detection of low concentrations of organisms e.g., 15 cells per g Source: and

11 © Copyright by Amplyus LLC, all rights reserved In the news

12 © Copyright by Amplyus LLC, all rights reserved How biotechnology can help fliC locus (non-pathogenic ) fliC fliC locus (pathogenic O157:H7 ) XmnI site Pathogenic E.coli gene differs from non-pathogenic E.coli Single nucleotide polymorphism (SNP) in fliC gene e.g. AAATTT changes to AAGTTT The SNP creates a restriction site in the fliC gene in E.coli O157:H7 SNP

13 © Copyright by Amplyus LLC, all rights reserved fliC Lab plan Template DNA PCR amplification Gel electrophoresis Meat plant A non-pathogenic Meat plant B O157:H7 Restriction digest 400bp bp fliC XmnI site

14 © Copyright by Amplyus LLC, all rights reserved 1.Template DNA to be amplified 2.Pair of DNA primers 3.DNA polymerase 4.dNTPs 5.Buffer to maintain pH and provide Mg 2+ 6.Thermal cycler What goes in a PCR reaction FWD primer REV primer Taq A A A A A A A T T T T G T T C C C C C C C G G G G G G G

15 © Copyright by Amplyus LLC, all rights reserved A.DNA sample from meat packing B.plant “A” B.DNA sample from meat packing plant “B” D.Control DNA from non-pathogenic E.coli H.Control DNA from pathogenic I.E.coli Setting up your PCR reactions Label 4 PCR tubes per group Tube “A”Tube “B” Tube “P”Tube “NP” FIELD SAMPLES USDA “REFERENCE LAB”

16 © Copyright by Amplyus LLC, all rights reserved Prepare 4 PCR tubes (200µL tubes) DNA sample A 15 µL 10 µL 5 µL A DNA sample B B Control P DNA P Control NP DNA NP 2X EZ PCR Master mix (EZ PCR Master Mix: PCR Buffer + Mg 2+ + Taq + dNTPs) 3X Food Safety Primer mix (Forward and Reverse primers) Also add your initials to side of tube

17 © Copyright by Amplyus LLC, all rights reserved Programming PCR parameters Initial denaturation:94°C 30 seconds Denaturation:94°C 5 seconds Annealing57°C 5 seconds Extension72°C 5 seconds x25 cycles (if prioritizing speed) OR x30 cycles (if prioritizing robustness of results) Final extension72°C 30 seconds

18 © Copyright by Amplyus LLC, all rights reserved Monitoring PCR amplification What is happening to DNA molecules at each step? Denaturation Annealing Extension Why do we need to add an enzyme (Taq polymerase)? What temperature is optimal for most enzymes? What makes Taq unique? How many more molecules of DNA will we have with each PCR cycle? And at the end of the entire PCR reaction? We call this exponential amplification How will we know which meat processing plant is behind the outbreak? Which caveats should the USDA investigators consider?

19 © Copyright by Amplyus LLC, all rights reserved Quiz: Which of these are NOT characteristics of PCR primers?  A. Short synthetic oligonucleotide  B. Typically bases in length  C. Double stranded DNA  D. Unique homology to the DNA template  E. Sequence with ~50% G:C content

20 © Copyright by Amplyus LLC, all rights reserved Set up restriction digest (XmnI) PCR product ABPNP 15 µL AXBXPXNPX Restriction digest (incubate at 37°C) 1 µL Restriction enzyme (XmnI restriction endonuclease) +

21 © Copyright by Amplyus LLC, all rights reserved Incubate 10 minutes at 37°C

22 © Copyright by Amplyus LLC, all rights reserved Next step: visualize the DNA fragments amplified by PCR 1. Pour an agarose gel2. Load the PCR products 4. Visualization in a transilluminator3. Electrophoresis e- - Pole + Pole

23 © Copyright by Amplyus LLC, all rights reserved Load Agarose Gel Load the gel as follows: 1.10µL DNA Ladder 2.12µL PCR product A 3.12µL PCR Product B 4.12µL PCR Product P 5.12µL PCR Product NP 6.12µL Restriction Digest AX 7.12µL Restriction Digest BX 8.12µL Restriction Digest PX 9.12µL Restriction Digest NPX

24 © Copyright by Amplyus LLC, all rights reserved Questions to probe deeper – (After gel run) How did the investigation turn out? Which plant might be the source of the outbreak? Why? What’s the importance of running controls from the USDA Reference Lab? What caveats should be applied when analyzing these results? What was the most unexpected thing you learned?

25 © Copyright by Amplyus LLC, all rights reserved Thank you We hope you enjoyed this lab!

26 © Copyright by Amplyus LLC, all rights reserved Additional resources Outbreak Detection Since Jack in the Box: A Public Health Evolution a-public-health-evolution/#.VNre1J3F-Sp CDC PulseNet Home Centers for Disease Control: E.coli outbreaks Popular books around E.coli O157:H7 outbreaks Toxin (by Robin Cook): Poisoned (by Jeff Benedict): Americans/dp/ X

27 © Copyright by Amplyus LLC, all rights reserved Appendix: Potential variants of this lab Variant 1: Food safety inspectors & USDA Reference Lab Food Safety Inspectors: Process DNA from Plant A and Plant B USDA Reference Lab: Process control DNA (P and NP) Variant 2: Testing more than 2 plants Most lab groups receive NP DNA Selected 1-2 groups receive P DNA

28 © Copyright by Amplyus LLC, all rights reserved Appendix: Copy cycles amplify DNA exponentially

29 © Copyright by Amplyus LLC, all rights reserved How miniPCR TM thermal cyclers enable DNA amplification Heated lid Prevents condensation 2 2. Heating block Separates DNA strands, preparing them for copy 3. Cooling fans Cools DNA, priming it for copy 4. Microprocessor Stores and controls temperature cycles