Download Crime Lab Classroom Slides

DNA amplification and analysis:
miniPCRTM Crime Lab
Release v4.1: October 2016
© Copyright by Amplyus LLC, all rights reserved
Welcome!
Our goals for today:
► Review DNA structure and DNA amplification concepts
► Solve a crime mystery using DNA analysis!
PCR is at the heart of DNA analysis
Molecular
diagnostics
Text
Consumer
genomics
Personalized
medicine
Text
Text
PCR
Food and
agriculture
Text
Text
Text
Forensics
Human
evolution
DNA analysis in personal ID
DNA
extraction
DNA
amplification
DNA
visualization
What other techniques could you use?
Let’s do it – CSI style!
Micropipetting
PCR
45 min – miniPCR™
Gel
electrophoresis
DNA
visualization
45 min – blueGel™
Content slide
miniPCR Crime Lab:
Missy Baker Missing!
► Engaging scenario
► Experimentally robust
► Confidence-building
► CFTR biology
► Core biotech skills
► PCR
► Gel electrophoresis
Missy Baker has a deletion mutation
CFTR1 primer
Healthy gene
(most people)
CFTR Gene
CFTR2 primer
Healthy person: the amplified DNA fragment always has the same
size, corresponding to the distance between the PCR primers
CFTR1 primer
Deletion mutation
(very rare)
Deletion
Short PCR fragment
mutated CFTR
GeneGene
CFTR
CFTR2 primer
Missy Baker!
Person carrying a CFTR deletion: the amplified DNA fragment from
the mutated gene is smaller than in most (healthy) individuals
CFTR:
Cystic Fibrosis Transmembrane Regulator
Note: STRs most often used in Personal ID
Other forms of genetic variation can also establish identity
Source of genetic variation
Useful for
VNTRs
• 6-100 nucleotides
repeated in tandem
• Bone marrow
transplants
STRs
• 2-6 nucleotides
repeated in tandem
• Forensics
• Disease marker
Structural
• Deletion, insertion,
duplication (e.g. in
CFTR gene)
SNPs
• Single nucleotide
polymorphisms
• Disease marker,
ancestry tracing
miniPCR in personal ID
STR analysis using capillary electrophoresis
Data used with permission of Signature Science LLC
Download: miniPCR Case
Study in Forensic Science
Warming up:
Micropipetting practice
Micropipetting practice
1. Identify volume range
2. Adjust volume
3. Press plunger to FIRST STOP
4. Press plunger to SECOND
STOP
Now, transfer real liquids: 20, 10, and 5µl
1. Adjust volume
2. Get a tip
3. Press plunger to FIRST STOP
4. Collect liquid. Release plunger
5. Transfer liquid
6. Press plunger to SECOND
STOP
7. Eject tip
Experimental set up
FORENSIC SAMPLES
Tube A
A. Hair DNA samples
found in
Suspect A car
Reference lab controls
Tube H
H. Control DNA
from healthy
CFTR gene
Tube B
B.
Hair DNA samples
found in
Suspect B car
Tube D
D. Control DNA
from CFTR
deletion mutant
What goes in a PCR experiment
1. Template DNA to be amplified
2. Pair of DNA primers
Taq
3. DNA polymerase
CFTR1 primer
CFTR2 primer
4. dNTPs
G
5. Buffer to maintain pH
and provide Mg2+
A
A
C TG
G
TA
C
C
Let’s set up 4 PCR tubes
2X EZ PCR Master Mix, Load Ready™
15 µL
(EZ PCR Master Mix: PCR Buffer + Mg2+ + Taq + dNTPs)
3X Crime Lab Primer mix
10 µL
5 µL
(Forward and Reverse primers)
DNA from
Suspect A’s
car
DNA from
Suspect B’s
car
Control DNA
healthy
CFTR gene
Control DNA
CFTR
deletion
A
B
H
D
Also add your initials to side of tube
PCR parameters
Initial denaturation:
94°C
30 seconds
Denaturation:
Annealing
Extension
94°C
57°C
72°C
5 seconds
5 seconds
8 seconds
x30 cycles
Final extension
72°C
30 seconds
PCR: Polymerase Chain Reaction
Find and replicate a specific DNA target
Complex DNA
sample
Region of
interest
Amplified DNA
(~1B copies)
How PCR works: 3 steps to copy DNA
1
94°C
Denaturation
2
50-60°C
Primer 1
Annealing
3
Extension
Primer 2
72°C
Taq DNA polymerase
dNTPs
Thermal cycling:
repeated temperature changes
denatured DNA
Single molecule
DNA + primers
DNA + copy
94° C
~1B copies
72° C
50-60° C
Denaturation
Annealing
Extension
Repeat x 30 cycles
How does each component enable PCR?
1
2
3
4
Discussion: observe miniPCR cycling
• 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 molecules of DNA will we have after each PCR cycle?
• And at the end of the entire PCR reaction?
• We call this exponential amplification
Reminder:
PCR relies on DNA’s unique structure
DNA: a double helix...
Source: US National Library of Medicine, NIH, Thinkquest
...held together by base complementarity
Let’s think about Cystic Fibrosis
►
What is the subcellular location of the CFTR protein?
Integral membrane protein (transmembrane protein)
►
How many different types of CFTR mutations can cause cystic fibrosis?
Over 1,300 mutations known
►
How common do you think CFTR mutations are? Are they dominant?
Recessive?
Most common recessive disorder in Caucasians. 1/25 people is a carrier.
►
What would we see if Missy Baker were heterozygous for the CFTR
deletion?
►
What types of treatments can help people with CF?
CFTR mutations affect water
balance in the lung airway lining
Outside of
epithelial lining
(lung airway)
H2O molecules enter lung
mucus by osmosis, hydrating it
Mucus becomes too
sticky, difficult to clear,
and traps bacteria
Healthy CFTR
protein
Inside of lung
epithelial cell
CFTR absent or defective,
affecting Chloride transport
Chloride
ions
Lung airway of
unaffected person
Lung airway of person
with Cystic Fibrosis
Effects of Cystic Fibrosis mutations
Cystic Fibrosis can be caused by 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.
Medically relevant CFTR mutations
Next step: Gel Electrophoresis
1. Pour agarose gel
2. Load PCR products
3. Electrophoresis
4. Visualization in a transilluminator
- Pole
e+ Pole
blueGel™: integrated
electrophoresis AND visualization system
Cast 2% agarose gels (per gel):
• 20 ml 1X TBE buffer
• 0.4 g agarose
45 sec in microwave
• Cool for ~1 min
• Add 2 µl GreenView Plus
DNA Stain
A
Load 15 µl of
PCR product
B
H
D
Ladder
Predict results
Missy Baker has a ~400 bp deletion in CFTR
Size in base pairs
H
D
Wrap up
► 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 other controls could we have run?
► What caveats apply when analyzing DNA evidence?
Expected results
Thank you!
www.miniPCR.com
[email protected]
@miniPCR
Facebook.com/miniPCR
Appendix: additional resources
Interpreting CFTR mutations
► http://ghr.nlm.nih.gov/gene/CFTR
► http://www.cftrscience.com/
The Innocence Project
► http://www.innocenceproject.org/
History of the use of DNA in crime solving
► http://www.forensicmag.com/articles/2005/01/evolution-dna-evidence-crime-solvingjudicial-and-legislative-history
Deutsche Welle: 30 years of DNA Fingerprinting
► http://www.dw.de/from-paternity-to-criminal-cases-dna-fingerprinting-has-been-30years-of-eureka/a-17911987
STRs are the most commonly analyzed
genetic polymorphism in forensics
STR analysis
►
Extract genomic DNA
►
Amplify STRs by PCR
►
Separate by gel or capillary electrophoresis
Gold standard in personal ID
► Easier to preserve, amplify, and separate than VNTRs
► 13 STR loci serve as standard for FBI CODIS.
► Convicted felon DNA databases
► Mass disasters
► Paternity testing
What other forms of genetic variation could
be useful for personal ID?
VNTRs
Variable Number Tandem Repeats
• Locus where DNA sequence is
organized as a tandem repeat
• Core repeat from 6-100 bp; 1-20
kb alleles
• Found on many chromosomes,
usually in subtelomeric regions
• Each variant acts as an inherited
allele allowing use for
identification
• Useful in genetics, biology
research, forensics and DNA
fingerprinting
STRs
Short Tandem Repeats
• Pattern of TWO or more
nucleotides repeated in tandem
• Repeat unit typically 2-6 bp; 50300 bp allele
• Typically found in introns,
>10,000 published human STRs
• Number of repeats at each
locus can create unique genetic
profile
• Prevalent method for
determining genetic profiles in
forensic cases
The complete biotech toolkit:
DNA Discovery System™
Micropipetting
PCR
Gel
electrophoresis
Visualization