- GenoSensor Corporation
GenoSensor Corporation
EduPrimer™ VNTR DNA Profiling Kit
Catalog # 3003
Version B
June 2015
User Manual
EduPrimer™ VNTR DNA Profiling Kit Manual
Table of Contents
Notes for Instructors ................................................................................ 2
Shipping, Storage and Safety .................................................................. 3
Suggestions for Teachers ........................................................................ 4
EduPrimer™ VNTR DNA Profiling Kit Overview ..................................... 5
Kit Components and Storage Conditions .................................................................................................. 5
Additional Required Materials .................................................................................................................. 5
Introduction: Theory and Background .................................................... 6
EduPrimer™ VNTR DNA Profiling Kit: Protocol ..................................... 7
Preparation ............................................................................................................................................... 7
DNA Preparation ....................................................................................................................................... 7
PCR Reaction ............................................................................................................................................. 7
Agarose Gel Electrophoresis ..................................................................................................................... 9
Results and Discussion ............................................................................................................................ 10
EduPrimer™ DNA Profiling Kit: Background ....................................... 11
Troubleshooting ..................................................................................... 15
Technical Service ................................................................................... 16
Literature Citation
When describing a procedure for publication using these products, please refer to them as the
EduPrimer™ VNTR DNA Profiling Kit.
Trademarks
EduPrimer™ is a trademark of GenoSensor.
Notes for Instructors
Kit Components and Storage Conditions:
Component
Solution A
Solution B
Cotton Swabs
2X PCR Master Mix
Positive Control DNA
Negative Control (DNase- RNase-free H2O)
DNA ladder
Storage
Room temp.
Room temp.
Room temp.
-20ºC
-20ºC
-20ºC
-20ºC
Preparation for DNA isolation and PCR for up to 24 students
Set heat block or water bath to 90ºC
Instruct the students in cheek cell collection step to make sure proper technique and timing is
used to ensure sufficient collection for DNA isolation.
Thaw 2X PCR Master Mix on ice. Before opening tube, spin 10 sec at 6,000 rpm or greater in a
microcentrifuge. Vortex 10 seconds, then spin again for 10 seconds.
Aliquot the 2X PCR Master Mix as necessary after doing the above preparation.
Each package contains enough 2X PCR Master Mix for 30 reactions. Use 10 ul of 2X PCR
Master Mix with 10 ul isolated DNA for a final PCR volume of 20 ul.
Include materials for 3 positive and 3 negative controls (10 ul from Control tube + 10 ul 2X PCR
Master Mix for one reaction).
Electrophoresis
Electrophoresis reagents are not provided in the kit. Please refer to the required materials list.
Best results are obtained by adding DNA dye (i.e., Gel Red,Sybr Safe) to molten agarose.
Avoid exposing the agarose gel to light, if possible perform gel polymerization and
electrophoresis in the dark.
DNA ladder supplied is enough for 3 lanes with 10 uL each.
Positive control supplied is enough for 6 lanes with 10 uL each after PCR.
Negative control supplied is enough for 6 lanes with 10 uL each after PCR.
After PCR, load up to 20 ul of student PCR reaction into a lane.
Shipping, Storage and Safety
Shipping and Storage
EduPrimer™ VNTR DNA Profiling kits are shipped on blue ice. Components should be
stored at temperatures shown in the above table. At proper storage conditions, components are
stable for 1 year from the date received. Expiration dates are also noted on product labels.
Safety Warnings and Precautions
This product is intended for research use only. It is not recommended or intended for the
diagnosis of disease in humans or animals. Do not use internally or externally in humans or
animals. Consider all chemicals as potentially hazardous. Only persons trained in laboratory
techniques and familiar with the principles of good laboratory practice should handle these
products. Wear suitable protective clothing such as laboratory overalls, safety glasses, and
gloves. Exercise caution to avoid contact with skin or eyes: if contact should occur, wash
immediately with water (Safety Data Sheets for products are available upon request).
Suggestions for Teachers
This kit is technical in nature and an excellent tool for teaching techniques, and has real links to
DNA Forensics. Students will enjoy extracting their own DNA and seeing their individual band
separation. Should you want to, there are other ways to spice things up even more. Here are a
few possible suggestions:
Smooth Criminal
1. Secretly pick two students to be the criminal. This can be done by collecting all of the student
supernatant DNA samples after students are done with them and selecting two of the tubes
from the whole class. Make note of which student’s samples you selected. Alternatively, collect
swabs from the “criminal” students’ cheek cells ahead of the class period and have them
prepared alongside the rest of the class.
2. Run the “criminal” samples through the PCR process alongside the rest of the class.
3. Run the criminal samples on the same gel (or gels) as the rest of the class for easy
comparison. Each PCR reaction can be split into two gels, with a 20 µl PCR product split in half
into two gels.
4. Have the students compare bands and identify the "criminals."
EduPrimer™ VNTR DNA Profiling Kit Overview
The EduPrimer™ VNTR DNA Profiling Kit introduces Polymerase Chain Reaction (PCR)
techniques to students, or anyone wishing to learn PCR and its uses. It contains all reagents
necessary for DNA isolation and PCR. PCR is an important and valuable skill to have in
contemporary biological and related sciences. In this kit, the experiment generates varying
results from person to person, demonstrating the basis for the process of creating DNA profiles
that are used to differentiate one person from another. After completing this experiment,
students will be able to proficiently perform PCR and understand the concepts behind it.
Kit Components and Storage Conditions
(for a lab of 24 students)
Component
Solution A
Solution B
Cotton Swabs
2X PCR Master Mix
Positive Control DNA
Amount (30 rxn’s)
6 mL
0.6 mL
28
300 µL
Storage
Room temp.
Room temp.
Room temp.
-20ºC
30 µL
-20ºC
Negative Control
DNA ladder
30 µL
30 µL
-20ºC
-20ºC
Additional Required Materials















Thermal Cycler
Heat Block or (heat plate, Beaker with de-ionized water; water bath, Tube floater;
Thermometer)
Microcentrifuge
Microcentrifuge tubes
Vortexer (optional)
Micropipettes (p10, p100)
Pipette tips
PCR tubes (1/reaction)
Tube Racks
Ethanol or ethanol wipes
Electrophoresis equipment
Electrophoresis supplies: agarose, TBE buffer, , gel dye (eg., Sybr safe, Gel Red)
Scissors and tweezers
UV light box or “Gel Doc” equipment and program
Permanent marker
Introduction: Theory and Background
We are very different from each other on many levels, but but not as much as you might think
on the genetic level. In fact, a large portion of the genetic makeup from one person to the next
may differ by as little as 0.1%! Our genome consists of over 3 billion base pairs, and that 0.1%
still makes a big difference. The differences between genomes underlies the theory behind DNA
profiling. There are a number of specific regions on our genomes that reliably vary between
individuals. For this experiment, the focus will be on a “Variable Number Tandem Repeat
(NVTR)” region. Throughout the genome there are segments that feature small repeating
sequences of DNA. A repeated sequence is generally the same between individuals, but the
number of times it repeats can vary. Analyze enough of the VNTR segments, and a a genetic
“fingerprint” for an individual can be generated.
In the EduPrimer VNTR DNA Profiling kit, we focus on the Penta E “microsatellite” region, a
VNTR region with a repeating sequence 2-5 base pairs long, which features a sequence of
“AAAGA” that can repeat anywhere from 5 to 26 times. This means that when a region flanking
and including this repeating sequence is amplified by PCR using our primers the region will be
between 246 – 351 base pairs long. Each individual will have two bands on the final gel, one
inherited from each of their parents, apart from individuals who might have identical repeating
numbered sequences from both parents.
For a single VNTR site there can be a wide degree of variation and it is unlikely that any two
non-related persons will share the same band pattern. In a real forensic analysis using DNA
profiling or fingerprinting, many variable regions are used to accurately distinguish one
individual.
EduPrimer™ VNTR DNA Profiling Kit: Protocol
Preparation
1. Set heat block or water bath to 90 ºC. For a heat block, it is recommended to add water
or sand to ensure proper heat transfer. For a water bath, be sure tubes are tightly sealed
and not fully submerged to avoid contamination.
2. Thaw 2X PCR Master Mix on ice. Before opening tube, spin 10 sec at 6,000 rpm or
greater in a microcentrifuge. Vortex 10 seconds, then spin again for 10 seconds.
DNA Preparation
*Do not eat or brush teeth one hour prior to cheek cell collection. Wear gloves and
handle solutions carefully*
1. Add 200µl of Solution A (red label) to a marked microcentrifuge tube.
2. To collect cheek cells, thoroughly roll provided cotton swab inside cheek for at least 10
seconds. .
3. Place swab into marked tube with Solution A.
4. Cut swab stem so it will fit inside the tube. Make certain the cap will shut tightly.
5. Vortex for at least 10 seconds. Solution A contains components which chemically disrupt
cell membranes and begin to unravel proteins. Under these conditions, the cheek cells
will begin to lyse or break open, spilling cell contents into the solution in the tube.
6. Place sample in heat block to incubate at 90 °C for 5 minutes. Immediately place tube in
ice until ready for next step.This process continues to destroy proteins, particularly,
those that damage DNA.
7. Load sample into a mini or microcentrifuge taking care that there is another sample
directly across from your sample to keep the centrifuge in balance as it spins. Close
internal and external centrifuge lids.
8. Spin briefly (~10 seconds) to pool condensation that has collected on the cap.
9. Remove and dispose of swab using forceps (rinse forceps between each sample to
prevent contamination).
10. Add 20 µl Solution B (green label) to the sample tube. Solution B neutralizes the harsh
conditions needed for lysis, preparing the solution for DNA isolation and PCR to follow.
11. Close the tube lid tightly and vortex to mix for at least 10 seconds.
12. Load sample into microcentrifuge with the tube hinge pointing out, balancing out your
sample tube, and closing the lids.
13. Spin sample for 1 minute at 12,000 rpm.
14. Look for a small clear round pellet near the bottom of the tube under the hinge. The
pellet contains cellular debris. The aqueous solution (supernatant) that has not
precipitated into the pellet contains cellular DNA.
PCR Reaction
*Wear gloves and handle solutions carefully*
1. Program the thermocycler as indicated below.
2. Prepare and label the top and side of a small PCR tube with your name
3. Ensure that the “2X PCR Master Mix” is on ice and has been spun at 6,000 RPM or
greater in a microcentrifuge for 10 seconds, vortexed for 10 seconds, then spun again
for 10 seconds before opening the Master Mix tube.
4. Label and prepare PCR tubes for controls (as directed by instructor) following the table
below
5. Add 10 µl of “2X PCR Master Mix” and 10 µl of supernatant from step 14 above (avoid
the pellet) to the labeled PCR tube for a total of 20 µl as indicated in the table below. The
supernatant contains your genomic DNA for polymerase chain reaction (PCR.)(Note: It is
preferred that the PCR reaction mix preparation is done on ice).
6. Mix the 20 µl PCR reaction mixture by pipetting in and out with the pipette, and the close
the lid tightly.
7. Store the sample on ice until it is ready to be loaded into the thermal cycler.
PCR reaction mixture tables
Student Sample PCR Mixture
2x PCR Master Mix
10 µl
Genomic Template (Supernatant)
10 µl
Volume total
20 µl
Control Samples PCR Mixture
Positive Control
2x PCR Master Mix
Negative control
10 µl 2x PCR Master Mix
10 µl
Positive control DNA
10 µl Negative Control
10 µl
Volume total
20 µl Volume total
20 µl
The positive control will have widely spaced bands near the upper and lower limits of this VNTR
region. The negative control does not contain DNA.
PCR Parameters
Program your thermal cycler as follows:
1.
2.
3.
4.
5.
94ºC – 2 minutes
94ºC denaturing – 20 seconds}
65ºC annealing – 20 seconds} repeat steps 2, 3, & 4 for 40 cycles
68ºC extension – 30 seconds}
68ºC – 5 minutes
6. 4ºC – finished / hold
Agarose Gel Electrophoresis
General Procedure, detailed directions as given by instructor

Prepare 2 – 2.5% agarose.

Set up electrophoresis apparatus and pour the 2 – 2.5% % molten agarose for gelation.

For staining, use a DNA dye which is added directly to the molten agarose. For light
sensitive dyes, keep the gel in the dark during gelation, either by performing in a dark
room or placing a box over the gel.

Use at least 10 µL of PCR product to visualize results by electrophoresis on agarose gel.
If gel well volume will accommodate more than 10 ul, a higher volume is preferred.
Loading dye was added to the mastermix to ensure that the sample will sink to the
bottom of the well and properly enter the agarose gel.

Run at ~100V for ~20 minutes and stop before loading dye has run off gel. Depending
on the DNA dye used, caution may need to be taken to reduce exposure of gel to light.

Visualize under UV and record the results manually or by photography

Compare individual experimental bands to positive control DNA. There are different DNA
sizes and most of them are between 240 – 360 bp. Positive control is 87 bp (monitoring
PCR performance).
Example of Gel Setup and Loading: Run 3 gels with 12 wells each to accommodate 24
students and 6 control reactions.
Lane 1:
10 µl DNA Ladder
Lane 2:
10 µl Negative Control
Lane 3:
10 µl Positive Control
Lanes 4 -12
Up to 20 µl of each student sample
Results and Discussion
Observe and record your results. Include a
diagrammed picture/photo of your bands.
Compare band pattern with the other students' band
patterns. Describe the similarities and differences.
Summarize the process of PCR using correct
terminology.
Describe a new experiment you could perform using
PCR and DNA gel electrophoresis.
EduPrimer™ DNA Profiling Kit: Background
Introduction to PCR
In 1983, during his time working at Cetus Corporation, Kary Mullis developed a
technique that significantly changed the field of genetics and all other biological sciences.. This
revolutionary process was termed “polymerase chain reaction,” or PCR, and he earned the
Nobel Prize in Chemistry in 1993 due to his innovation. His technique enabled all
researchersnot just a few expert microbiologists, to amplify DNA. Before that, amplification of
DNA was extremely difficult and time consuming. Now, scientists in any field can incorporate
molecular biology into their research with PCR.
Currently, PCR is used in a wide variety of areas including: gene mapping, DNA
sequencing, gene expression, gene detection, forensics, criminal investigation, medical
diagnostics, and genome sequencing. Very few of these applications were practically possible
before PCR. The process does require an initial investment in specialized machinery, but with
the proper equipment, nearly anyone can perform a successful PCR without significant cost.
PCR and Biotechnology — Revolutionizes an Entire Research Community
PCR is capable of producing large quantities of targeted DNA from a very small amount
of starting material, known as the template. DNA can be obtained from nearly any cell i.e.,
blood cells, hair cells, cheek cells, etc., and after proper treatment to isolate the DNA, PCR can
be applied to create millions of copies of nearly any desired DNA sequence. The power of PCR
is its specificity; using primers to target a known sequence, it will amplify only that segment of
DNA even though the entire genome was placed in the reaction tube.
The basic components of PCR:
- Reaction Buffer
- DNA nucleotides (dNTP’s) of each adenine, guanine, thymine and cytosine
- DNA polymerase
- Forward and reverse DNA oligonucleotide primers
- Template DNA (starting material)
PCR Makes Use of Two Basic Processes in Molecular Genetics
1. Complementary DNA strand hybridization
For DNA to be amplified, one must have a known sequence which flanks the gene of
interest upstream and downstream. These sequences are used to create ‘oligonucleotide
primers,’ meaning a short ~20 base pair nucleotide sequence which is used as a starting point
for DNA replication. The primers are complementary to their target regions so they will anneal
(attach) to those regions specifically. Primers are required because DNA polymerase cannot
add nucleotides without a preexisting chain.
Complementary strand hybridization occurs when two different oligonucleotide primers
anneal to each of their respective complementary base pair sequences on the template. They
are designed specifically to anneal at opposite ends of opposite strands of the specific
sequence of DNA that is desired to be amplified
2. DNA strand synthesis via DNA polymerase
In a PCR, a special type of DNA polymerase is used that is able to withstand the
temperature fluctuations required for thermal cycling. Most DNA polymerases cannot tolerate
the high temperatures and fluctuations from ~60ºC-94ºC. The breakthrough in PCR came with
the isolation of DNA polymerase from a thermophilic bacterium known as as Thermus
aquaticus. This bacterial species lives in high temperature steam vents and therefore its DNA
polymerase evolved to withstand high temperatures.
During PCR, DNA is synthesized and doubles each cycle, thus the growth of DNA copy
# over the reaction is exponential. In theory, after 30 cycles there will be 230 copies ; over a
billion copies of DNA. Yielding this much DNA allows the for visualization through a variety of
means. One of the most popular visualization methods is agarose gel electrophoresis.
Genes and DNA
The human genome contains 23 pairs of chromosomes that contain a total of
thirty to fifty thousand genes, most of which code for proteins. However, those genes only
comprise about 5% of the genome, leaving 95% as so-called non-coding DNA. This noncoding
DNA is found not only between, but within genes, splitting them into segments. In eukaryotes,
non-coding DNA sequences found within genes are known as introns. The sequences that do
code for proteins are called exons. In eukaryotes, genomic DNA is transcribed into RNA
molecules containing both introns and exons for a particular gene. While the RNA is still in the
nucleus (before being transported out of the nucleus), the introns (in = stay within the nucleus)
must be removed from the RNA while the exons (ex = exit the nucleus) are spliced together to
form the complete coding sequence which will soon be translated into the protein. This process
is called RNA splicing. Some genes may contain a few introns, others may contain dozens.
Interestingly, it is the non-coding ‘junk’ DNA that is useful to us when considering the DNA
profile of an individual, instead of the DNA that actually codes for life.
As discussed, functional segments of genes (exons) code for proteins. Proteins are
molecules that carry out most cellular functions. Exon sequences are therefore very similar
among individuals, because even a slight difference can change the function of the protein in a
potentially harmful way (many diseases are caused by mutated proteins). Introns, however,
often vary in size and number among individuals. Intron sequences are thought to be the result
of the differential accumulation of mutations throughout evolution that are silently passed to
descendants through the genetic code. It is this difference in intron sequences that allows us to
determine human genetic diversity. The identification of these distinctive characteristics in DNA
represents the molecular basis for human identification and population genetics. Throughout
evolution, intron sequences have been the target of random insertions by short repetitive
interspersed elements (SINEs). SINEs have become randomly inserted within our introns over
millions of years.
One such repetitive element is called the Alu element (Figure 1). The Alu element is a
DNA sequence about 300 base pairs long that is repeated; one copy at a time, almost 500,000
times within the human genome. The origin and function of such randomly repeated sequences
is not yet known. The Alu name comes from the Alu I restriction enzyme (enzymes that cut DNA
at specific sequences) recognition site that is found in this sequence.
PCR Stages
The machinery required to perform PCR is known as a thermal cycler. The thermal
cycler enables the steps of PCR to be automated. The reaction involves a repetitive series of
cycles, each of which consists of template denaturation, primer annealing, and extension of the
annealed primer by Taq DNA polymerase. Before beginning DNA amplification, genomic DNA is
prepared from students' cells. The students’ DNA is added to a mixture of reagents:
oligonucleotide primers, thermo-stable DNA polymerase (Taq), the four nucleotides (A, T, G, C),
and reaction buffer. These reagents are pre-mixed as a 2X PCR Master Mix in the EduPrimer™
DNA profiling kit. The tubes are placed into the thermal cycler contains an aluminum block that
holds the samples and can be rapidly heated and cooled across extreme temperature
differences. The rapid heating and cooling of this thermal block is called temperature cycling or
thermal cycling.
The first step of the PCR temperature cycling procedure heats the sample to
94°Ccausing the template strands separate. This is called the denaturation step.
The thermal cycler then rapidly cools to 60°C allowing the primers to anneal to the
separated template strands. This is called the annealing step. The two original template strands
may re-anneal to each other or compete with the primers for the primers complementary binding
sites. However, the primers are added in excess such that the primers actually out-compete the
original DNA strands for the primers’ complementary binding sites.
Lastly, the thermal cycler heats the sample to 72°C for Taq DNA polymerase to extend
the primers and make complementary DNA strands of the target sequence. This is called the
extension step. to make The two new sets of double-stranded DNA (dsDNAwill be used for
another cycle and subsequent strand synthesis. At this stage, a complete temperature cycle
(thermal cycle) has been completed.
Each step takes 30 seconds to 1 minute, and will repeat for 30-40 cycles depending on how the
user has programmed the thermal cycler. At the end of the cycles, the product is put on hold
at 4°C until the user is ready to proceed to the analysis of the product.
Figure 3. Experiment flowchart from start to finish
Troubleshooting
Symptom
Possible causes
Solutions
No amplification
product
Questionable template quality
Analyze starting material
Inhibitory Substance in reaction
Decrease sample volume
Insufficient cycle #
Run additional cycles
Incorrect thermal cycler program
Verify times and temperatures
Errors in heat block incubation
Calibrate heating block, use
sand or water to maximize
contact with tube for proper
heat transfer
Autoclave tubes and use filter
tips
Lower annealing temperature
in 2ºC increments
Increase swabbing time,
thoroughly swab.
Contaminated tubes/solutions
Primer annealing temperature too high
Weak bands/faint
signal
Low concentration of DNA template
DNA Dye degradation during
preparation
Expired, contaminated or degraded
DNA dye
Non-specific
amplification product
Premature Taq polymerase replication
Primer annealing temperature too low
Insufficient mixing of reaction solution
Exogenous DNA contamination
Light sensitive dyes should be
kept in the dark during gel
preparation. Prepare in dark
room or place a box over the
electrophoresis apparatus
during gelation and
electrophoresis.
Verify that the DNA dye has
not degraded in storage, been
contaminated or expired.
Mix solutions on ice, place rxn
directly into 94º thermal cycler
Raise annealing temperature
in 2ºC increments
Mix solutions thoroughly
before beginning the reaction
-Wear gloves
-Use dedicated area for
sample preparation
-Use non-aerosol tips
Technical Service
For more information or technical assistance, please call, write, fax, or email.
GenoSensor Corporation
4665 S. Ash Avenue
Suite G-18
Tempe, Arizona 85282
Tel: 1-480-598-5378
Fax: 1-480-755-3319
Email: [email protected]
Web: www.genosensorcorp.com
Limited Warranty
GenoSensor is committed to providing our customers with high-quality goods and services. Our goal is to
ensure that every customer is 100% satisfied with our products and our service. If you should have any
questions or concerns about a GenoSensor product or service, please contact our Technical Service at
[email protected] GenoSensor warrants that all of its products will perform according
to the specifications stated on the certificate of analysis. This warranty limits GenoSensor Corporation’s
liability only to the cost of the product. No warranty is granted for products beyond their listed expiration
date. No warranty is applicable unless all product components are stored in accordance with instructions.
GenoSensor reserves the right to select the method(s) used to analyze a product unless GenoSensor
agrees to a specified method in writing prior to acceptance of the order. GenoSensor makes every effort
to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is
inevitable. Therefore GenoSensor makes no warranty of any kind regarding the contents of any
publications or documentation. If you discover an error in any of our publications, please report it to our
Technical Service. GenoSensor assumes no responsibility or liability for any special, incidental, indirect or
consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other
warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a
particular purpose
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement