release v2.4: january 2015 © copyright by amplyus llc, all rights reserved dna amplification and...

Release v2.4: January 2015© Copyright by Amplyus LLC, all rights reserved

DNA amplification and analysis:miniPCRTM Crime Lab

Science for everyone, everywhere

2© Copyright 2014-5 by Amplyus LLC, all rights reserved

Welcome!

Our goals for today

1. Review DNA structure and DNA amplification concepts

2. Solve a crime mystery using PCR!


PCR is at the heart of DNA analysis

PCR

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Forensics

Food and agriculture

Consumer genomics

Molecular diagnostics

Personalized medicine

Human evolution

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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 ofinterest

Genetic testing

Pathogen detection

Drug development

Crop modification

Forensic analysis

Sequencing

Etc.

Applications


PCR relies on DNA’s unique structure

Source: US National Library of Medicine, NIH, Thinkquest

DNA: a double helix...DNA: a double helix......held together by base

complementarity...held together by base

complementarity


How PCR works: 3 steps to copy DNA

Denaturation

1 94°C

Annealing

250-60°C

Primer 2

Primer 1

Extension

372°C

Taq DNA polymerase dNTPs


How PCR works: repeat the cycle

Denaturation

Annealing Extension

DNA + primers

denatured DNA

DNA + copy

94° C

50-60° C

72° C

Repeat x ~25-30 cycles

Single molecule ~1B copies


PCR makes DNA visible (and useful)


PCR experimentation chain

Set-up VisualizationPCRElectrophores

is


How miniPCRTM enables DNA amplification

1

3

4

1. Heated lid• Prevents

condensation22. 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


Today, we will use DNA analysis to solve a Crime Mystery

PCR (DNAamplification)

Gel electrophoresis

DNA visualization


Use of DNA in personal identification


Missy Baker Gone Missing!

FreeBoston metroMissy Baker, pastry shop owner goes missing,

police at a lossThe mysterious disappearance of the baker raises serious concerns within the population. An enigma that befuddles police investigators.

With two abduction suspects in custody, local students volunteer to try to find the missing baker.

Boston, October 20th 2015. Husband Ned reported Missy "Sugar-Cup" Baker missing, fretful after not finding her at the shop following his daily nap. The couple resides in the apartment above the pastry shop at 2 Middleborough Rd. a popular fixture in this usually quiet neighborhood. Anxiety takes hold in the community. Quickly following the report of the missing baker (wheat-blond and thin as a stick) investigators identified two suspects, but both have remained silent after harsh interrogation.

Wooly mammoth p.5

finally clonedTransgenic kiwi p.7

solves world hunger

Following extensive police searches, investigators found blond hair strands in each of their cars.

A godsend for science students, who volunteer with high-tech DNA analysis equipment to identify the kidnapper.


CFTR protein: Cystic Fibrosis Transmembrane Receptor Gene


How to find Missy Baker: mutation in her CFTR gene

CFTR Gene

CFTR1 primer

CFTR2 primer

Healthy person: the amplified DNA fragment always has the same size, corresponding to the distance between the PCR primers

Healthy gene(most people)

CFTR1 primer

CFTR2 primer

Person carrying a CFTR deletion: the amplified DNA fragment from the mutated gene is smaller than in most (healthy) individuals

Deletion mutation(very rare)

Short PCR fragment CFTR Gene mutated CFTR Gene Deletion


How to interpret PCR results (example)

Siz

e in

bas

e p

airs


1. Template DNA to be amplified

2. Pair of DNA primers

3. DNA polymerase

4. dNTPs

5. Buffer to maintain pH and provide Mg2+

6. Thermal cycler

What goes in a PCR reaction

CFTR1 primer

CFTR2 primer

Taq

AA

A

A AA

A

TT

T

T

G

T

T

C

C

C

C

C

C

C G

GG

GG

G G


Prepare 4 PCR tubes (200µL tubes)

DNA sample

from hair found with Suspect A

15 µL

10 µL

5 µL

A

DNA sample

from hair found with Suspect B

B

Control DNA from

healthy CFTR gene

H

Control DNA

from CFTR deletion mutant

D

PCR Master mix(NEB One Taq: PCR Buffer + Mg2+ + Taq + dNTPs)

Primer mix(Forward and Reverse primers)

Also add your initials to side of tube


Alternative approach: 2 PCR Tubes per groupLabel 2 PCR tubes per group

A. Hair DNA sample from suspect A’s car

Tube “A” Tube “B”

“DETECTIVES”

Groups 1 through 4

B. Hair DNA samplefrom suspect B’s car

“REFERENCE LAB”

Groups 5 through 8

Tube “H” Tube “D”

D. Control DNA from CFTR deletion mutant

H. Control DNA from healthy CFTR gene


Programming PCR parameters

Initial denaturation: 94°C 30 seconds

Denaturation: 94°C 5 secondsAnnealing 57°C 5 secondsExtension 72°C 10 seconds

x30 cycles

Final extension 72°C 30 seconds


Programming

1

2

3

30 5 5 10 30

57.0


Monitoring DNA amplification

What is happening to DNA molecules at each PCR 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


Quiz: Which of these are NOT characteristics of PCR primers?

A. Short synthetic oligonucleotide

B. Typically 18-25 bases in length

C. Double stranded DNA

D. Unique homology to the DNA template

E. Sequence with ~50% G:C content


Next step: visualize the DNA fragments amplified by PCR

1. Pour an agarose gel 2. Load the PCR products

4. Visualization in a transilluminator

3. Electrophoresis

e-

- Pole

+ Pole


Questions to probe deeper – I (During gel run)

What is the subcellular location of the CFTR gene product?

How many different types of CFTR mutations can cause cystic fibrosis?

How common do you think CFTR mutations are?

Are these mutations typically dominant? Recessive?

What would we see if Missy Baker were heterozygous for the CFTR deletion?

What can be done to treat people with CFTR gene mutations?


Medically relevant CFTR mutations


CFTR mutations affect water balance in the lung airway lining


What are the effects of Cystic Fibrosis (CF) genetic mutations?

Cystic Fibrosis is due to one or more mutations in the CFTR gene, which encodes a channel protein involved in the passage of chloride ions through the cell membrane.

The defective gene interferes with the body’s ability to transfer water and salt to and from cells. This causes secretions, which are normally thin and watery in healthy people, to become very thick and sticky.

The thick secretions clog up organs and prevent them from working properly. An increase in the viscosity of cell secretions often causes respiratory diseases, which are the main cause of death from CF.


Questions to probe deeper – II (After gel run)

How do you think the investigation turned out?

Is the evidence consistent with suspect A being guilty? Why?

Is the evidence consistent with suspect B being innocent? Why?

What’s the importance of running controls from the Reference Lab?

What caveats should be applied when analyzing forensic DNA evidence?

What was the most unexpected thing you learned today?


Thank you

We hope you enjoyed this lab!


Additional resources

Interpreting CFTR mutationshttp://ghr.nlm.nih.gov/gene/CFTRhttp://www.cftrscience.com/

The Innocence Projecthttp://www.innocenceproject.org/

History of the use of DNA in crime solvinghttp://www.forensicmag.com/articles/2005/01/evolution-dna-evidence-crime-solving-judicial-and-legislative-history

Deutsche Welle: 30 years of DNA Fingerprintinghttp://www.dw.de/from-paternity-to-criminal-cases-dna-fingerprinting-has-been-30-years-of-eureka/a-17911987

http://ghr.nlm.nih.gov/gene/CFTR

http://ghr.nlm.nih.gov/gene/CFTR

http://www.cftrscience.com/

http://www.innocenceproject.org/



http://www.forensicmag.com/articles/2005/01/evolution-dna-evidence-crime-solving-judicial-and-legislative-history




http://www.dw.de/from-paternity-to-criminal-cases-dna-fingerprinting-has-been-30-years-of-eureka/a-17911987

http://www.dw.de/from-paternity-to-criminal-cases-dna-fingerprinting-has-been-30-years-of-eureka/a-17911987


Appendix: PCR cycles amplify DNA exponentially

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