Student Manual

Introduction to PCR — The Polymerase Chain Reaction

You are about to perform a procedure known as PCR1 to amplify a specific sequence of
your own DNA in a test tube. You will be looking for a particular piece of DNA that is present
in the genes of many, but not all, people. Analysis of the data generated in this laboratory
will enable you to determine whether or not you carry this specific DNA sequence.
The genome, composed of DNA, is our hereditary code. This is the so-called
blueprint that controls much of our appearance, behavior, and tendencies. Molecular
biology is the study of genes and the molecular details that regulate the flow of genetic
information from DNA to RNA to proteins, from generation to generation. Biotechnology
uses this knowledge to manipulate organisms’ (microbes, plants, or animals) DNA to help
solve human problems.
Within the molecular framework of biology, DNA, RNA, and proteins are closely tied to
each other. Because proteins and enzymes ultimately play such a critical role in the life
process, scientists have spent many lifetimes studying proteins in an attempt to understand
how they work. With this understanding, it was believed we could cure, prevent, and
overcome disease and physical handicaps as well as explain exactly how and why
organisms exist, propagate, and die. However, the complete answer to how and why does
not lie solely in the knowledge of how enzymes function; we must learn how they are
made. If each enzyme is different, then what controls these differences and what is the
blueprint for this difference? That answer lies within our genome, or genetic code.
Thus, you may realize why researchers today, in an attempt to understand the
mechanisms behind the various biological processes, study nucleic acids as well as proteins
to get a complete picture. In the last 20 years, many advances in nucleic acid techniques
have allowed researchers to study the roles that nucleic acids play in biology. It took the
imagination and hard work of many scientists to reveal the answers to one of the most
mysterious puzzles of life — understanding the mechanisms that control how DNA is
translated into proteins within living cells.

Before Beginning This Lab, See If You Can Answer the Following
Questions
How is DNA faithfully passed on from generation to generation? What causes genetic
disease in some people but not others? How do scientists obtain DNA to study? What
secrets can DNA tell us about our origins? What human problems can an understanding of
DNA help us solve? Should we unlock the secrets held in this most basic building block of
life?

PCR Set the Stage for a Scientific Revolution

In 1983, Kary Mullis2 at Cetus Corporation developed the molecular biology technique
that has since revolutionized genetic research. This technique, called the polymerase
chain reaction (PCR), transformed molecular biology into a multidisciplinary research field
within 5 years of its invention. Before PCR, the molecular biology techniques used to study
DNA required such a high level of expertise that relatively few scientists could use them.
The objective of PCR is to produce a large amount of DNA in a test tube (in vitro),
starting from only a trace amount. Technically speaking, this means the controlled enzymatic
amplification of a DNA sequence, or gene, of interest. The template strands can be any
form of DNA, such as genomic DNA. A researcher can use tiny amounts of genomic DNA

39
from a drop of blood, a single hair follicle, or a cheek cell, and make enough DNA to study.
In theory, only a single template strand is needed to copy and generate millions of new
identical DNA molecules. Prior to PCR, this would have been impossible. It is the ability to
amplify the precise sequence of DNA of interest that is the true power of PCR.
PCR has made an impact on four main areas of genetic research: gene mapping;
cloning; DNA sequencing; and gene detection. PCR is now used as a medical diagnostic
tool to detect specific mutations that may cause genetic disease;3 in criminal investigations
and courts of law to identify suspects,4 and in the sequencing of the human genome.5 Prior
to PCR, the use of molecular biology techniques for therapeutic, forensic, pharmaceutical,
agricultural, or medical diagnostic purposes was neither practical nor cost-effective. The
development of PCR transformed molecular biology from a difficult science to one of the
most accessible and widely used disciplines of biotechnology.
Two methods for DNA template preparation are provided in the manual. Your instructor
will indicate which exercise to follow. Now, let’s extract some of your own DNA.

40
Lesson 1 Cheek Cell DNA Template Preparation
To obtain DNA for use in the polymerase chain reaction (PCR) you will extract the DNA
from your own living cells. It is interesting to note that DNA can be also extracted from
mummies and fossilized dinosaur bones. In this lab activity, you will isolate DNA from
epithelial cells that line the inside of your cheek. To do this, you will rinse your mouth with a
saline (salt) solution, and collect the cells using a centrifuge. You will then boil the cells to
rupture them and release the DNA they contain. To obtain pure DNA for PCR, you will use
the following procedure:
The cheek cells are transferred to a microcentrifuge tube containing InstaGene™
matrix. This particulate matrix is made up of negatively charged, microscopic beads that
chelate, or grab, metal ions out of solution. It traps metal ions, such as Mg2+, which are
required as catalysts or cofactors in enzymatic reactions. Your cheek cells will then be
lysed, or ruptured, by heating to release all of their cellular constituents, including enzymes
that were once
contained in the cheek-cell lysosomes. Lysosomes are sacs in the cytoplasm that contain
powerful enzymes, such as DNases, which are used by cells to digest the DNA of invading
viruses. When you rupture the cells, these DNases can digest the released DNA. However,
when the cells are lysed in the presence of the chelating beads, the cofactors are adsorbed
and are not available to the enzymes. This virtually blocks enzymatic degradation of the
extracted DNA so you can use it as the template in your PCR reaction.
You will first suspend your isolated cheek cells in the InstaGene matrix and incubate
them at 56°C for 10 minutes. This preincubation step helps to soften plasma membranes
and release clumps of cells from each other. The heat also inactivates enzymes, such as
DNases, which can degrade the DNA template. After this 10 minute incubation period,
place the cells in a boiling (100°C) water bath for 5 minutes. Boiling ruptures the cells and
releases DNA from their nuclei. You will use the extracted genomic DNA as the target
template for PCR amplification.

41
Lesson 1 Cheek Cell DNA Template Preparation (Lab Protocol)
Workstation Checklist
Materials and supplies required at the workstations prior to beginning this exercise are
listed below.

Student Workstation Quantity per Station (✔)

1.5 ml microcentrifuge tubes 4 ❐
Screwcap tubes with 200 µl InstaGene matrix 4 ❐
2–20 µl adjustable-volume micropipet 1 ❐
Pipet tips (filter type), 2–20 µl 4 ❐
Permanent marker 1 ❐
Copy of Quick Guide or protocol 1 ❐
Waste container 1 ❐
Cups with 10 ml 0.9% saline 4 ❐

Instructor’s Workstation Quantity per Class

100–1,000 µl adjustable-volume pipet 1 ❐
Pipet tips (filter type), 100–1,000 µl 1 box ❐
56°C and 100°C water baths or dry baths 1 each ❐
Microcentrifuge 1 ❐
or mini centrifuge 4 ❐
Vortexer (optional) 1 ❐

42
Lesson 1 Cheek Cell DNA Template Preparation (Lab Protocol)
1. Each member of your team should have 1 screwcap tube containing 200 µl
InstaGene™ matrix, 1.5 ml microcentrifuge tube, and a cup containing 10 ml of 0.9%
saline solution. Label one of each tube and a cup with your initials.

2. Do not throw away the saline after completing this step. Pour the saline from the cup
into your mouth. Rinse vigorously for 30 seconds. Expel the saline back into the cup.

3. Set a 100–1,000 µl micropipet to 1,000 µl and transfer 1 ml of your oral rinse into the
microcentrifuge tube with your initials. If no 100–1,000 µl micropipet is available, care-
fully pour ~1 ml of your swished saline into the microcentrifuge tube (use the markings
on the side of the microcentrifuge tube to estimate 1 ml).

4. Spin your tube in a balanced centrifuge for 2 minutes at full speed. When the centrifuge
has completely stopped, remove your tube. You should be able to see a pellet of
whitish cells at the bottom of the tube. Ideally, the pellet should be about the size of a
match head. If you can’t see your pellet, or your pellet is too small, pour off the saline
supernatant, add more of your saline rinse, and spin again.

Centrifuge

5. Pour off the supernatant and discard. Taking care not to lose your cell pellet, carefully
blot your microcentrifuge tube on a tissue or paper towel. It’s OK for a small amount
of saline (~50 µl, about the same size as your pellet) to remain in the bottom of the
tube.

6. Resuspend the pellet thoroughly by vortexing or flicking the tubes until no cell clumps
remain.

43
7. Using an adjustable volume micropipet set to 20 µl, transfer your resuspended cells into
the screwcap tube containing the InstaGene with your initials. You may need to use the
pipet a few times to transfer all of your cells.
8. Screw the caps tightly on the tubes. Shake or vortex to mix the contents.
9. When all members of your team have collected their samples, incubate the tubes for 10
min in a 56°C water bath or dry bath. At the halfway point (5 minutes), shake or vortex
the tubes several times. Place the tubes back in the water bath or dry bath for the
remaining 5 minutes.

Water bath or
dry bath 56°C, 10 min

10. Remove the tubes from the water bath or dry bath and shake them several times.
Incubate the tubes for 5 min at 100°C in a water bath (boiling) or dry bath for 5 minutes.

Water bath or
100°C, 5 min
dry bath

11. Remove the tubes from the 100°C water bath or dry bath and shake or vortex several
times to resuspend the sample. Place the eight microcentrifuge tubes in a balanced
arrangement in a centrifuge. Pellet the matrix by spinning for 5 minutes at 6,000 x g (or
10 minutes at 2,000 x g).

Centrifuge

12. Store your screwcap tube in the refrigerator until the next laboratory period, or proceed
to step 2 of Lesson 2 if your teacher instructs you to do so.

44
Lesson 1 Hair Follicle DNA Template Preparation
To obtain DNA for use in the polymerase chain reaction (PCR) you will extract the DNA
from your own living cells. It is interesting to note that DNA can be also extracted from
mummies and fossilized dinosaur bones. In this lab activity, you will isolate DNA from the
epithelial cells that coat the base of your hair. To do this, you will collect two hairs from
yourself. Good places to obtain hair are your head, arm, or leg. You will then boil the cells
to lyse, or rupture them and release the DNA they contain. To obtain pure DNA for PCR
you will use the following procedure:
You will trim 2 hairs with an obvious sheath (a coating of cells surrounding the base of
the hair) or a large root (the bulb-shaped structure at the base of the hair) to about 2 cm
then transfer them into a microcentrifuge tube containing InstaGene matrix and protease.
This particulate matrix is made up of negatively charged microscopic beads that chelate, or
grab, metal ions out of solution. InstaGene traps metal ions, such as Mg+2, which are
required as catalysts or cofactors in enzymatic reactions. Protease digests the connections
between cells, allowing better lysis in the next step. Your epitheleal cells will then be lysed,
or ruptured, by heating to release all of their cellular constituents, including enzymes that
were once contained in the epitheleal cell lysosomes. Lysosomes are sacs within the
cytoplasm that contain powerful enzymes, such as DNases, which are used by cells to
digest the DNA of invading viruses. When you rupture the cells, these DNases can digest
the released DNA. However, when the cells are lysed in the presence of the InstaGene
beads, the cofactors are adsorbed and are not available to the enzymes. This virtually
blocks enzymatic degradation of the extracted DNA so you can ust it as the template in
your PCR reaction.
You first suspend isolated hair-follicle cells in the InstaGene matrix with protease and
incubate them at 56°C for 10 minutes. This preincubation step helps to soften plasma
membranes and release clumps of cells from each other and the hair. After this 10 minute
incubation period, place the cells in a water bath at 100°C (boiling) or dry bath for 5
minutes. Boiling ruptures the cells and releases DNA from cell nuclei. The heat also
inactivates enzymes such as DNases, which can degrade the DNA template. You will use
the extracted genomic DNA as the target template for PCR amplification.

45
Lesson 1 Hair Follicle DNA Template Preparation (Lab Protocol)
Workstation Checklist
Materials and supplies required at the workstation prior to beginning this exercise are
listed below.

Student Workstations Quantity per Station (✔)

Screwcap tubes with InstaGene matrix plus protease 4 ❐
Permanent marker 1 ❐
Waste container 1 ❐
Copy of Quick Guide or protocol 1 ❐
Tweezers or forceps 1 ❐
Scissors or razor blade 1 ❐
Instructor’s workstation Quantity per Class
56°C and 100°C water baths or dry baths 1 of each ❐
Microcentrifuge 1 ❐
or mini centrifuge 4 ❐
Vortexer (optional) 1 ❐

46
Lesson 1 Hair Follicle DNA Template Preparation (Lab Protocol)
1. Each member of your team should have 1 screwcap tube containing 200 µl of InstaGene
matrix plus protease. Label the tube on the cap and side with your initials.

2. Collect 2 hairs from yourself. Choose hairs that leave noticeable sheaths (a coating of
epitheleal cells around the base of the hair). Alternatively, choose hairs that have a
large root. The root is the bulb-shaped base of the hair. Keeping the end of the hair with
the sheath and bulb, trim the hair with scissors so it is ~2 cm long. Place your trimmed
hairs into the screwcap tube with your initials. Screw the cap tightly on the tube.

Hair

Sheath
Bulb

3. Incubate the tubes at 56°C for 10 minutes in a water bath or dry bath. At the halfway
point (5 minutes), shake or vortex the tube gently, then place it back in the 56°C water
bath or dry bath for the remaining 5 minutes.

Water bath or
dry bath 56°C, 10 min

4. Remove your tube, gently shake or vortex it, then place it in a boiling water bath or dry
bath set at 100°C for 5 minutes.

Water bath or
dry bath 100°C, 5 min

47
5. Remove your tube from the boiling water bath and shake or vortex to resuspend the
contents. Pellet the matrix by spinning at 6,000 x g for 5 minutes (or 2,000 x g for 10
minutes).

Centrifuge

6. Store your screwcap tube in the refrigerator until the next laboratory period (or proceed
to step 2 of Lesson 2).

48
Lesson 1 DNA Template Preparation

Focus Questions
1. Why is it necessary to chelate the metal ions from solution during the boiling/lysis step
at 100°C? What would happen if you did not use a chelating agent such as the
InstaGene matrix?

2. What is needed from the cells for PCR?

3. What structures must be broken to release the DNA from a cell?

4. Why do you think the DNA is stored cold with the InstaGene matrix after boiling the
samples?

49
Lesson 2 PCR Amplification
It is estimated that there are 30,000–50,000 individual genes in the human genome.
The true power of PCR is the ability to target and make millions of copies of (or amplify) a
specific piece of DNA (or gene) out of a complete genome. In this activity, you will amplify a
region within your chromosome 16.
The recipe for a PCR amplification of DNA contains a simple mixture of ingredients. To
replicate a piece of DNA, the reaction mixture requires the following components:
1. DNA template — containing the intact sequence of DNA to be amplified
2. Individual deoxynucleotides (A, T, G, and C) — raw material of DNA
3. DNA polymerase — an enzyme that assembles the nucleotides into a new DNA chain
4. Magnesium ions — a cofactor (catalyst) required by DNA polymerase to create the
DNA chain
5. Oligonucleotide primers — pieces of DNA complementary to the template that tell DNA
polymerase exactly where to start making copies
6. Salt buffer — provides the optimum ionic environment and pH for the PCR reaction
The template DNA in this exercise is genomic DNA that was extracted from your cells.
The complete master mix contains Taq DNA polymerase, deoxynucleotides, oligonu-
cleotide primers, magnesium ions, and buffer. When all the other components are com-
bined under the right conditions, a copy of the original double-stranded template DNA
molecule is made — doubling the number of template strands. Each time this cycle is
repeated, copies are made from copies and the number of template strands doubles —
from 2 to 4 to 8 to 16 and so on — until after 20 cycles there are 1,048,576 exact copies of
the target sequence.
PCR makes use of the same basic processes that cells use to duplicate their
DNA.
1. Complementary DNA strand hybridization
2. DNA strand synthesis via DNA polymerase
The two DNA primers provided in this kit are designed to flank a DNA sequence within
your genome and thus provide the exact start signal for the DNA polymerase to “zero in on”
and begin synthesizing (replicating) copies of that target DNA. Complementary strand
hybridization takes place when the two different primers anneal, or bind to each of their
respective complementary base sequences on the template DNA.
The primers are two short single-stranded DNA molecules (23 bases long), one that is
complementary to a portion of one strand of the template, and another that is
complementary to a portion of the opposite strand. These primers anneal to the separated
template strands and serve as starting points for DNA Taq replication by DNA polymerase.
Taq DNA polymerase extends the annealed primers by “reading” the template strand and
synthesizing the complementary sequence. In this way, Taq polymerase replicates the two
template DNA strands. This polymerase was isolated from a heat-stable bacterium (Thermus
aquaticus) which in nature lives within high temperature steam vents such as those found in
Yellowstone National Park.6 For this reason these enzymes have evolved to withstand high
temperatures (94°C) and can be used in the PCR reaction.

50
PCR Step by Step
PCR amplification includes three main steps, a denaturation step, an annealing step,
and an extension step (summarized in Figure 9). In denaturation, the reaction mixture is
heated to 94°C for 1 minute, which results in the melting or separation of the double-stranded
DNA template into two single stranded molecules. PCR amplification, DNA templates must
be separated before the polymerase can generate a new copy. The high temperature
required to melt the DNA strands normally would destroy the activity of most enzymes, but
Taq polymerase is stable and active at high temperature.

3'
5'
5'
3'

Denature strands at 94°C

5' 3'

3' 5'

Anneal primers at 60°C

(Taq polymerase recognizes 3' ends
of primers)

5' 3'
Primer 3' 5'
Taq polymerase
5' 3' Primer

3' 5'

Extend at 72°C
(Synthesize new strand)

5' 3'

3' 5'

5' 3'

3' 5'

Repeat cycle 40 times

Fig. 9. A complete cycle of PCR.

During the annealing step, the oligonucleotide primers “anneal to” or find their
complementary sequences on the two single-stranded template strands of DNA. In these
annealed positions, they can act as primers for Taq DNA polymerase. They are called
primers because they “prime” the synthesis of a new strand by providing a short sequence
of double-stranded DNA for Taq polymerase to extend from and build a new complementary
strand. Binding of the primers to their template sequences is also highly dependent on
temperature. In this lab exercise, a 60°C annealing temperature is optimum for primer
binding.
During the extension step, the job of Taq DNA polymerase is to add nucleotides (A, T, G,
and C) one at a time to the primer to create a complementary copy of the DNA template. During

51
polymerization the reaction temperature is 72°C, the temperature that produces optimal Taq
polymerase activity. The three steps of denaturation, annealing, and extension form one “cycle”
of PCR. A complete PCR amplification undergoes 40 cycles.
The entire 40 cycle reaction is carried out in a test tube that has been placed into a
thermal cycler. The thermal cycler contains an aluminum block that holds the samples and
can be rapidly heated and cooled across broad temperature differences. The rapid heating
and cooling of this thermal block is known as temperature cycling or thermal cycling.
Temperature Cycle = Denaturation Step (94°C) + Annealing Step (60°C) + Extension Step (72°C)

52
Lesson 2 PCR Amplification (Lab Protocol)

Workstation Checklist
Materials and supplies that should be present at the workstations prior to beginning this
lab are listed below.

Student Workstation Quantity per Station (✔)

PCR tubes 4 ❐
PCR capless adapter tubes 4 ❐
Complete master mix (with primers) on ice 1 tube ❐
2–20 µl adjustable-volume micropipet 1 ❐
Pipet tips (filter type), 2–20 µl 8 ❐
Ice bucket with ice 1 ❐
Permanent marker 1 ❐
Waste containers 1 ❐
Copy of Quick Guide or protocol 1 ❐

Instructor’s (common) workstation Quantity per Class

Gel trays 1 per 2 stations ❐
Molten agarose 40 ml per gel ❐
Lab tape for gel trays 1 per station ❐
Thermal cycler 1 ❐
Microcentrifuge 1 ❐
or mini centrifuge 4 ❐

53
Lesson 2 PCR Amplification (Lab Protocol)

1. Obtain your screwcap tube that contains your genomic DNA template from the
refrigerator. Centrifuge your tubes for 2 minutes at 6,000 x g or for 5 minutes at
2,000 x g in a centrifuge.

2. Each member of the team should obtain a PCR tube and capless microcentrifuge tube.
Label each PCR tube on the side of the tube with your initials and place the PCR tube
into the capless microcentrifuge tube as shown.

PCR tube Capless

tube

3. Transfer 20 µl of your DNA template from the supernatant in your screwcap tube into
the bottom of the PCR tube. Do not transfer any of the matrix beads into the PCR
reaction because they will inhibit the PCR reaction.

Supernatant

Matrix

DNA template PCR tube

4. Locate the tube of yellow PCR master mix (labeled “Master”) in your ice bucket.
Transfer 20 µl of the master mix into your PCR tube. Mix by pipetting up and down
2–3 times. Cap the PCR tube tightly and keep it on ice until instructed to proceed to the
next step. Avoid bubbles, especially in the bottom of the tubes.

Master mix

54
5. Remove your PCR tube from the PCR capless adapter tube and place the PCR tube in
the thermal cycler.

6. When all of the PCR samples are in the thermal cycler, the teacher will begin the PCR
reaction. The reaction will undergo 40 cycles of amplification, which will take
approximately 3 hours.

7. If your teacher instructs you to do so, you will now pour your agarose gels (the gels
may have been prepared ahead of time by the teacher).

55
Lesson 2 PCR Amplification

Focus Questions
1. Why is it necessary to have a primer on each side of the DNA segment to be amplified?

2. How did Taq DNA polymerase acquire its name?

3. Why are there nucleotides (A, T, G, and C) in the master mix? What are the other
components of the master mix, and what are their functions?

4. Describe the three main steps of each cycle of PCR amplification and what reactions
occur at each temperature.

5. Explain why the precise length target DNA sequence doesn’t get amplified until the
third cycle. You may need to use additional paper and a drawing to explain your
answer.

56
Lesson 3 Gel Electrophoresis of Amplified PCR Samples and
Staining of Agarose Gels
What Are You Looking At?
Before you analyze your PCR products, let’s take a look at the target sequence being
explored.

What Can Genes and DNA Tell Us?

It is estimated that the 23 pairs, or 46 chromosomes, of the human genome
(23 chromosomes come from the mother and the other 23 come from the father) contain
approximately 30,000–50,000 genes. Each chromosome contains a series of specific
genes. The larger chromosomes contain more DNA, and therefore more genes, compared
to the smaller chromosomes. Each of the homologous chromosome pairs contains similar
genes.
Each gene holds the code for a particular protein. Interestingly, the 30,000–50,000
genes only comprise 5% of the total chromosomal DNA. The other 95% is noncoding DNA.
This noncoding DNA is found not only between, but within genes, splitting them into
segments. The exact function of the noncoding DNA is not known, although it is thought
that noncoding DNA allows for the accumulation of mutations and variations in genomes.
When RNA is first transcribed from DNA, it contains both coding and noncoding
sequences. While the RNA is still in the nucleus, the noncodong introns (in = stay within
the nucleus) are removed from the RNA while the exons (ex = exit the nucleus) are
spliced together to form the complete messenger RNA coding sequence for the protein
(Figure 10). This process is called RNA splicing and is carried out by specialized
enzymes called spliceosomes.

Intron 1 Intron 2

5' Exon 1 Exon 2 Exon 3 3' Genomic DNA

Transcription

5' Exon 1 Exon 2 Exon 3 3' Pre-mRNA Genotype

Splicing

Exon 1 Exon 2 Exon 3 mRNA

Translation

Protein Phenotype

Fig. 10. Splicing of introns from genes.

Introns often vary in their size and sequence among individuals, while exons do not. This
variation is thought to be the result of the differential accumulation of mutations in DNA
throughout evolution. These mutations in our noncoding DNA are silently passed on to our
descendants; we do not notice them because they do not affect our phenotypes. However,
these differences in our DNA represent the molecular basis of DNA fingerprinting used in
human identification and studies in population genetics.

57
The Target Sequence
The human genome contains small, repetitive DNA elements or sequences that have
become randomly inserted into it over millions of years. One such repetitive element is
called the “Alu sequence”7 (Figure 11). This is a DNA sequence about 300 base pairs
long that is repeated almost 500,000 times throughout the human genome.8 The origin
and function of these repeated sequences is not yet known.

5' ALU 3'

Intron

Fig. 11. Location of an Alu repetitive element within an intron.

Some of these Alu sequences have characteristics that make them very useful to
geneticists. When present within introns of certain genes, they can either be associated
with a disease or be used to estimate relatedness among individuals. In this exercise,
analysis of a single Alu repeat is used to estimate its frequency in the population and as
a simple measure of molecular genetic variation — with no reference to disease or
relatedness among individuals.
In this laboratory activity you will look at an Alu element in the PV92 region of
chromosome 16. This particular Alu element is dimorphic, meaning that the element is
present in some individuals and not others. Some people have the insert in one copy of
chromosome 16 (one allele), others may have the insert in both copies of chromosome 16 (two
alleles), while some may not have the insert on either copy of the chromosome (Figure 12).
The presence or absence of this insert can be detected using PCR followed by agarose
gel electrophoresis.
Since you are amplifying a region of DNA contained within an intron, the region of DNA
is never really used in your body. So if you don’t have it, don’t worry.
The primers in this kit are designed to bracket a sequence within the PV92 region that
is 641 base pairs long if the intron does not contain the Alu insertion, or 941 base pairs long
if Alu is present. This increase in size is due to the 300 base pair sequence contributed by
the Alu insert.
When your PCR products are electrophoresed on an agarose gel, three distinct
outcomes are possible. If both chromosomes contain Alu inserts, each amplified PCR
product will be 941 base pairs long. On a gel they will migrate at the same speed so there
will be one band that corresponds to 941 base pairs. If neither chromosome contains the
insert, each amplified PCR product will be 641 base pairs and they will migrate as one
band that corresponds to 641 base pairs. If there is an Alu insert on one chromosome but
not the other, there will be one PCR product of 641 base pairs and one of 941 base pairs.
The gel will reveal two bands for such a sample.

58
 PV92 Genotype DNA Size of PCR Products

ALU

ALU Homozygous (+/+) 941 base pairs

Homozygous (–/–) 641 base pairs

ALU

Heterozygous (+/–) 941 and 641 base pairs

Fig. 12. The presence or absence of the Alu insert within the PV92 region of chromosome 16.

Electrophoresis separates DNA fragments according to their relative sizes. DNA

fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a
conductive buffer solution. A direct current is passed between wire electrodes at each
end of the chamber. DNA fragments are negatively charged, and when placed in an electric
field will be drawn toward the positive pole and repelled by the negative pole. The matrix of
the agarose gel acts as a molecular sieve through which smaller DNA fragments can move
more easily than larger ones. Over a period of time, smaller fragments will travel farther than
larger ones. Fragments of the same size stay together and migrate in what appears as a
single “band” of DNA in the gel. In the sample gel below (Figure 13), PCR-amplified bands
of 941 bp and 641 bp are separated based on their sizes.

1 2 3 4 5 6 7 8

(bp)

1,000
700
500

200
100

Fig. 13. Electrophoretic separation of DNA bands based on size. EZ Load DNA molecular mass ruler, which
contains 1,000 bp, 700 bp, 500 bp, 200 bp, and 100 bp fragments (lane 1); two homozygous (+/+) individuals with
941 bp fragments (lanes 2, 6); three homozygous (–/–) individuals with 641 bp fragments (lanes 3, 5, and 8), and
two heterozygous (+/–) individuals with 941 and 641 bp fragments (lanes 4 and 7).

59
Lesson 3 Gel Electrophoresis of Amplified PCR Samples and Staining
of Agarose Gels (Lab Protocol)

Workstation Checklist
Materials and supplies that should be present at the workstations prior to beginning this
lab are listed below.

Student workstation Quantity per Station (✔)

Agarose gel 1 ❐
Student PCR samples 1 per student ❐
MMR (DNA standard) 1 tube ❐
Orange G DNA loading dye 1 tube ❐
2–20 µl adjustable-volume micropipet 1 ❐
Pipet tips (filter type), 2–20 µl 12 ❐
Permanent marker 1 ❐
Gel box and power supply 1 ❐
Fast Blast™ DNA stain, 1x or 100x solution 120 ml per 2 stations ❐
Gel support film (optional) 1 ❐
Clear acetate sheets for tracing gels (optional) 1 ❐
Warm tap water for destaining gels (if performing 1.5–2 L per 2 stations ❐
quick staining protocol)
Large containers for destaining (if performing 1–3 per 2 stations ❐
quick staining protocol)
Copy of Quick Guide or protocol 1 ❐
Waste container 1 ❐

Instructor’s workstation Quantity per Class

1x TAE electrophoresis buffer 275 ml per gel box ❐
Amplified positive control samples (4 each) 12 ❐
PV92 homozygous (+/+)
PV92 homozygous (–/–)
PV92 heterozygous (+/–)
Shaking platform (optional)* 1 ❐
Microcentrifuge 1 ❐
or mini centrifuge 4 ❐

* Strongly recommended.

60
Lesson 3 Gel Electrophoresis of Amplified PCR Samples
(Lab Protocol)
1. Remove your PCR samples from the thermal cycler. If a centrifuge is available, place
the PCR tubes in the PCR capless adapter tubes and pulse-spin the tubes (~3 seconds
at 2,000 x g) to bring the condensation that formed on the lids to the bottom of the
tubes.
2. Add 10 µl of Orange G loading dye to each PCR tube and mix gently.
3. Obtain an agarose gel (either the one you poured or one pre-poured by your teacher).
Place the casting tray with the solidified gel in it, onto the platform in the gel box. The
wells should be at the cathode (–) end of the box, where the black lead is connected.
Very carefully remove the comb from the gel by pulling it straight up, slowly.
4. Pour ~275 ml of electrophoresis buffer into the electrophoresis chamber, until it just
covers the wells.

–
+

5. Using a clean tip for each sample, load the samples into the 8 wells of the gel in the
following order:

Lane Sample Load Volume

1 MMR (DNA standard) 10 µl
2 Homozygous (+/+) control 10 µl
3 Homozygous (–/–) control 10 µl
4 Heterozygous (+/–) control 10 µl
5 Student 1 20 µl
6 Student 2 20 µl
7 Student 3 20 µl
8 Student 4 20 µl

61
6. Secure the lid on the gel box. The lid will attach to the base in only one orientation: red
to red and black to black. Connect the electrical leads to the power supply.
7. Turn on the power supply. Set it to 100 V and electrophorese the samples for
30 minutes.

–
+

8. When electrophoresis is complete, turn off the power and remove the lid from the gel
box. Carefully remove the gel tray and the gel from the gel box. Be careful, the gel is
very slippery. Nudge the gel off the gel tray with your thumb and carefully slide it into
your plastic staining tray.

Staining of Agarose Gels

The moment of truth has arrived. What is your genotype? Are you homozygous or
heterozygous? To find out, you will have to stain your agarose gel. Since DNA is naturally
colorless, it is not immediately visible in the gel. Unaided visual examination of gel after
electrophoresis indicates only the positions of the loading dyes and not the positions of the
DNA fragments. DNA fragments are visualized by staining the gel with a blue dye called
Fast Blast DNA stain. The blue dye molecules are positively charged and have a high
affinity for the DNA. These blue dye molecules strongly bind to the DNA fragments and
allow DNA to become visible. These visible bands of DNA may then be traced, photographed,
sketched, or retained as a permanently dried gel for analysis.

Directions for Using Fast Blast DNA Stain

Below are two protocols for using Fast Blast DNA stain in the classroom. Use protocol 1
for quick staining of gels to visualize DNA bands in 12–15 minutes, and protocol 2 for
overnight staining. Depending on the amount of time available, your teacher will decide
which protocol to use. Two student teams will stain the gels per staining tray (you may want
to notch gel corners for identification). Mark staining trays with initials and class period
before beginning this activity.
WARNING
Although Fast Blast DNA stain is nontoxic and noncarcinogenic, latex or vinyl
gloves should be worn while handling the stain or stained gels to keep hands from
becoming stained blue. Lab coats or other protective clothing should be worn to
avoid staining clothes.

62
Protocol 1: Quick Staining of Agarose Gels in 100x Fast Blast DNA
Stain
This protocol allows quick visualization of DNA bands in agarose gels within
15 minutes. For quick staining, Fast Blast DNA stain (500x) should be diluted to a 100x
concentration. We recommend using 120 ml of 100x Fast Blast to stain two 7 x 7 cm or
7 x 10 cm agarose gels in individual staining trays provided in Bio-Rad’s education kits. If
alternative staining trays are used, add a sufficient volume of staining solution to completely
submerge the gels.
Following electrophoresis, agarose gels must be removed from their gel trays before
being placed in the staining solution. This is easily accomplished by holding the base of the
gel tray in one hand and gently pushing out the gel with the thumb of the other hand.
Because the gel is fragile, special attention must be given when handling it. We highly
recommend using a large spatula or other supportive surface to transfer the gel from one
container to another. Destaining requires the use of at least one large-volume container,
capable of holding at least 500 ml, at each student workstation. Each student team may
utilize separate washing containers for each wash step, or simply use a single container
that is emptied after each wash and refilled for the next wash.
1. Mark the staining tray with your initials and class period. You will stain 2 gels per
tray.
2. Stain gels (2–3 minutes)
Remove each gel from the gel tray and carefully slide it into the staining tray. Pour
approximately 120 ml of 100x stain into the staining tray. If necessary, add more 100x
stain to completely submerge the gels. Stain the gels for 2–3 minutes, but not for more than
3 minutes. Using a funnel, pour the 100x stain into a storage bottle and save it for future
use. The stain can be reused at least 7 times.

3. Rinse gels 2–3 minutes

Transfer the gels into a large container containing 500–700 ml of clean, warm (40–55°C)
tap water. Gently shake the gels in the water for ~10 seconds to rinse.

10 seconds

4. Wash gels
Transfer the gel into a large container with 500–700 ml of clean, warm tap water. Gently
rock or shake the gels on a rocking platform for 5 minutes. If no rocking platform is available,
move the gels gently in the water once every minute.

5 minutes

63
5. Wash gels
Perform a second wash as in step 4.

5 minutes

6. Record and analyze results

Examine the stained gels for expected DNA bands. The bands may appear fuzzy immediate-
ly after the second wash, but will begin to develop into sharper bands within 5–15 minutes
after the second wash. This is due to Fast Blast dye molecules migrating into the gel and
binding more tightly to the DNA molecules.
To obtain maximum contrast, additional washes in warm water may be necessary. Destain
to the desired level, but do not wash the gels in water overnight. If you cannot complete the
destaining in the allocated time, you may transfer the gel to 1x Fast Blast stain for overnight
staining. See Protocol 2.
a. Place your gel on a light background and record your results by making a diagram
as follows. Place a clear sheet of plastic sheet or acetate over the gel. With a
permanent marker, trace the wells and band patterns onto the plastic sheet to
make a replica picture of your gel. Remove the plastic sheet for later analysis.
Alternatively, gels can be photocopied on a yellow piece of transparent film for
optimal contrast.
b. With the help of your instructor, determine whether you are homozygous or
heterozygous for the Alu insertion. First look at the control samples and note the
migration patterns of the homozygous +/+, the homozygous –/–, and the
heterozygous +/– samples (also refer to the example on page 59). You may notice
that in the heterozygous sample the smaller 641 base pair band is more intense
than the larger 941 bp band. This difference is due to the fact that the smaller
fragment is amplified more efficiently than the larger fragment. Copies of the shorter
fragment can be made at a faster rate than the bigger fragment, so more copies of
the shorter fragment are created per cycle. Refer to pages 69–72 for more
information on how to analyze your data.

c. Dry the agarose gel as a permanent record of the experiment.

i. Trim away any unloaded lanes with a knife or razor blade. Cut your gel from
top to bottom to remove the lanes that you did not load samples into.

64
 ii. Place the gel directly upon the hydrophilic size of a piece of gel support film.
(Water will form beads on the hydrophobic side of a piece of gel support film.)
Center the gel on the film and remove bubbles that may form between the gel
and film. Place the film on a paper towel and let the gel dry in a well-ventilated
area, making sure to avoid direct exposure to light. As the gel dries it will bond
to the film but will not shrink. If left undisturbed on the support film, the gel will
dry completely at room temperature after 2–3 days. The result will be a flat,
transparent, and durable record for the experiment. Tape the dried gel into your
laboratory notebook.

Gel support film

Note: Avoid extended exposure of dried gels to direct light to prevent band fading.
However DNA bands will reappear if the dried gels are stored in the dark for 2–3
weeks after fading.

65
Protocol 2: Overnight Staining of Agarose Gels in 1x Fast Blast DNA
Stain
For overnight staining, Fast Blast DNA stain (500x) should be diluted to a 1x
concentration. We recommend using 120 ml of 1x Fast Blast to stain two 7 x 7 cm or 7 x 10 cm
agarose gels in individual staining trays provided in Bio-Rad’s education kits. If alternative
staining trays are used, add a sufficient volume of staining solution to completely submerge
the gels.
Following DNA electrophoresis, agarose gels must be removed from their gel trays
before being placed in the staining solution. This is easily accomplished by holding the
base of the gel tray in one hand and gently pushing out the gel with the thumb of the other
hand. Because the gel is fragile, special attention must be given when handling it.
1. Mark staining trays with your initials and class period. You will stain two gels per tray.
2. Stain gels (overnight)*
Pour 1x stain into a gel staining tray. Remove each gel from the gel tray and carefully slide it
into the staining tray containing the stain. If necessary, add more 1x staining solution to
completely submerge the gels. Place the staining tray on a rocking platform and agitate
overnight. If no rocking platform is available, agitate the gels staining tray a few times
during the staining period. You should begin to see DNA bands after 2 hours, but at least
8 hours of staining is recommended for complete visibility of stained bands.

Stain overnight
3. Analyze results
No destaining is required after staining with 1x Fast Blast. The gels can be analyzed
immediately after staining.
a. Place your gel on a light background and record your results by making a diagram
as follows. Place a clear sheet of plastic sheet or acetate over the gel. With a
permanent marker, trace the wells and band patterns onto the plastic sheet to
make a replica picture of your gel. Remove the plastic sheet for later analysis.
Alternatively, gels can be photocopied on a yellow piece of transparent film for
optimal contrast.

* Shake the gels gently and intermittently during overnight staining in 1x Fast Blast DNA stain; small
DNA fragments tend to diffuse without shaking.

66
 b. With the help of your instructor, determine whether you are homozygous or
heterozygous for the Alu insertion. First look at the control samples and note the
migration patterns of the homozygous +/+, the homozygous –/–, and the heterozy-
gous +/– samples (also refer to the example on page 59). You may notice that in
the heterozygous sample the smaller 641 base pair band is more intense than
the larger 941 bp band. This difference is due to the fact that the smaller fragment
is amplified more efficiently than the larger fragment. Copies of the shorter fragment
can be made at a faster rate than the bigger fragment, so more copies of the shorter
fragment are created per cycle. Refer to pages 69–72 for more information on how
to analyze your data.

c. Dry the agarose gel as a permanent record of the experiment.

i. Trim away any unloaded lanes with a knife or razor blade. Cut your gel from
top to bottom to remove the lanes that you did not load samples into.

ii. Place the gel directly upon the hydrophilic size of a piece of gel support film.
(Water will form beads on the hydrophobic side of a piece of gel support film.)
Center the gel on the film on a paper towel and let the gel dry in a well-ventilated
area, making sure to avoid direct exposure to light. As the gel dries it will bond
to the film but will not shrink. If left undisturbed on the support film, the gel will
dry completely at room temperature after 2–3 days. The result will be a flat,
transparent, and durable record for the experiment. Tape the dried gel into
your laboratory notebook.

Gel support film

Note: Avoid extended exposure of dried gels to direct light to prevent band fading.
However, DNA bands will reappear if the dried gels are stored in the dark for 2–3
weeks after fading.

67
Lesson 3 Gel Electrophoresis of Amplified PCR Samples

Focus Questions
1. Explain the difference between an intron and an exon.

2. Why do the two possible PCR products differ in size by 300 base pairs?

3. Explain how agarose electrophoresis separates DNA fragments. Why does a smaller
DNA fragment move faster than a larger one?

4. What kind of controls are run in this experiment? Why are they important? Could others
be used?

68
Lesson 4 Analysis and Interpretation of Results
If the overnight staining protocol was used to stain the gels, record your results and dry
your gels as described earlier.

Analysis
Compare your sample lanes with the control lanes using the DNA size marker as a
reference. Mark the location and size of your fragment or fragments. By comparing your
DNA migration pattern to the controls, determine whether you are homozygous +/+,
homozygous –/–, or heterozygous +/–. If your sample lane is blank, discuss the possible
reasons with your classmates and teacher.
Remember that the interpretation of this gel allows you to determine your genetic makeup
only at the locus (chromosomal location), being studied. There are three possible
genotypes for the Alu insert at the location you have amplified. For a class, determine
the number of individuals of each genotype: homozygous +/+, homozygous –/–, and
heterozygous +/–. Tally the class results in the table on page 70.
A major factor affecting the reliability of DNA fingerprinting evidence in forensics is
population genetics and genetic statistics. In humans, there are hundreds of loci, or DNA
segments, like Alu, that can be selected and used for fingerprinting analysis. Depending on
demographic factors such as ethnicity and geographic isolation, some populations show
much less variation in particular DNA segments than others. A lower degree of variation will
increase the odds of more than one individual having the same sequence. If 33% (1 out of
three individuals) of a given population has the same fingerprinting pattern for a certain
DNA segment, then little information will be obtained from using that segment alone to
identify an individual. In such a case, a positive result would only identify a person with 33%
accuracy.
In analyzing how incriminating the DNA evidence is, one needs to ask the question:
Statistically, how many people in a population have the same DNA pattern as that taken
from a crime scene: 1 in 1,000,000? 1 in 10,000? 1 in 10?
For a DNA fingerprint to identify a suspect in a criminal case or a father in a paternity
suit, accurate identification required not a 1 out of 3 (1/3) chance of a match in a population,
but closer to a 1 in 10 million (1/107) chance of a match. The frequency of a particular DNA
pattern turning up in a population drastically decreases when multiple DNA segments are
selected and amplified, rather than just one segment. For DNA fingerprinting to be
admissible as evidence in court, it must analyze 30 to 40 different DNA segments on
multiple chromosomes from the same person.
The Alu insert you have fingerprinted in this exercise has been used to study the
migration patterns of human populations over time.8 The data from these studies have
been published, and your class samples can be compared to the data collected from much
larger populations.

69
Lesson 4 Analysis and Interpretation of Results

Focus Questions
Remember that this Alu sequence is inserted into a noncoding region of the PV92 locus
on chromosome 16 and is not related to a particular disease, nor does it code for any
protein sequence. It is simply a sequence that can be used to study human genotypic
frequencies.
Because Alu repeats appear in the general population at random, the Alu insert in
chromosome 16 is very useful for the study of gene frequencies in localized human
populations. Theoretically, in some small, geographically isolated populations, all
individuals may be homozygous +/+. In others, the individuals may all be homozygous –/–.
In a “melting-pot” population, the three genotypes (+/+, +/–, –/–) may exist in equilibrium.
The frequencies of genotypes and alleles are basic characteristics that population
geneticists use to describe and analyze populations. The results you obtain in this exercise
provide a real-life opportunity to calculate genotypic and allelic frequencies of the Alu insert
in your class and to use the Hardy-Weinberg equation.
The results of the PCR reactions reveal your and your classmates’ genotypes: +/+, +/–,
and –/–. Knowing your genotypes, you can count up the alleles of your class “population”
and determine their frequencies. You can then compare the allelic and genotypic frequen-
cies of your class population to published reports of larger population sizes.9

1. What is your genotype for the Alu insert in your PV92 region?

2. What are the genotypic frequencies of +/+, +/–, and –/– in your class population? Fill in
the table below with your class data.

Table 1. Observed Genotypic Frequencies for the Class

Category Number Frequency (# of Genotypes/Total)
Homozygous (+/+)
Heterozygous (+/–)
Homozygous (–/–)
Total = = 1.00

70
 Allelic frequencies can be calculated from the numbers and frequencies of the genotypes
in the population. Population geneticists use the terms p and q to represent the frequencies
of the (+) and (–) alleles, respectively. Allele frequencies can be calculated from either the
numbers or the frequencies of the genotypes (since they are related to each other).

p = frequency of (+) allele = number of (+) alleles

total number of alleles (both + and –)

= 2(# of +/+ students) + 1(# of +/– students)

total number of alleles (both + and –)

= frequency of (+/+) students + ½ (frequency of (+/–) students)

q = frequency of (–) allele = number of (–) alleles

total number of alleles (both + and –)

= 2 (# of –/– students) + 1(# of +/– students)

total number of alleles (both + and –)

= frequency of (–/–) students + ½ (frequency of (+/–) students)

3. What is the frequency of each allele in your class sample? Fill in the table below with
your class data. Remember, a class of 32 students (N) will have a total of 64 (2N)
instances of each locus.

Table 2. Calculated Allelic Frequencies for the Class

Category Number Frequency
(+) alleles p =
(–) alleles q =
Total alleles = = 1.00

4. The following table presents data from a USA-wide random population study.
Table 3. Genotypic Frequencies for Alu in a USA Sample
Category Number Frequency
Homozygous (+/+) 2,422 0.24
Heterozygous (+/–) 5,528 0.55
Homozygous (–/–) 2,050 0.21
Total = 10,000 = 1.00

71
 Now, using the data above, calculate the allelic frequencies for the USA data as you did
for your class population in Table 2.
Table 4. Calculated Allelic Frequencies for USA
Category Number Frequency
(+) alleles p =
(–) alleles q =
Total alleles = = 1.00

5. How do your actual class data for genotypic and allelic frequencies compare with those
of the random sampling of the USA population? Would you expect them to match? What
reasons can you think of to explain the differences or similarities?

The Hardy-Weinberg equation, p2 + 2pq + q2 = 1, is one of the foundations of popula-

tion genetics. It is the algebraic expansion of (p + q)2 = 1, where p + q = 1. The equation
describes the frequencies of genotypes in a population that is at “genetic equilibrium”,
meaning that the frequencies are stable from generation to generation. The Hardy-Weinberg
theory states that, for a population to achieve this equilibrium, the population must be quite
large, the members must mate randomly and produce offspring with equal success, and
there must be no migration of individuals into or out of the population, or an excessive
mutation converting one allele to another. Given these conditions, and the allelic frequencies
p and q, the Hardy-Weinberg equation says that
p2 = the expected frequency of the (+/+) genotype in the population
2pq = the expected frequency of the (+/–) genotype in the population
q2 = the expected frequency of the (–/–) genotype in the population
It is important to understand that p2, 2pq, and q2 are expected, theoretical genotype
frequencies of a population under Hardy-Weinberg equilibrium conditions, and they may not
be realized in real-life population samples if one of the conditions is not met. These theoretical
frequencies are calculated using the observed values for p and q; they may or may not be
the same as the observed genotypic frequencies such as those shown in Table 1. If the
observed and expected genotypic frequencies are the same, this indicates that the popula-
tion is in Hardy-Weinberg genetic equilibrium.
6. Using the values for p and q that you calculated in Table 2 for your class population,
calculate p2, 2pq, and q2. Do they come out to be the same as the genotype frequencies
that you found in Table 1? If they do, your class resembles a Hardy-Weinberg genetic
equilibrium. If your observed (actual) genotype frequencies are not the same as the
expected values, what might be some of the reason(s) for the difference?
7. Using the values for p and q that you calculated in Table 4 for the USA population
sample, calculate p2, 2pq, and q2. Do they come out to be the same as the genotype
frequencies that you found in Table 3? Does this USA-wide sample suggest that the
population of the USA is in Hardy-Weinberg equilibrium?

72
Lesson 5 Analysis of Classroom Data Using Bioinformatics
Bioinformatics is a discipline that integrates mathematical, statistical, and computer
tools to collect and process biological data. Bioinformatics has become an important tool
in recent years for analyzing the extraordinarily large amount of biological information
that is being generated by researchers around the world. In Lesson 5, you will perform a
bioinformatics exercise to investigate the genotypic frequencies for the Alu polymorphism in
your class population and compare them with the genotypic frequencies of other
populations.
Following PCR amplification and electrophoresis of your samples, you will analyze your
experimental data to determine your genotypes for the Alu insertion within the PV92 locus
on chromosome 16. The classroom genotype data can then be entered into the Allele Server
database located at Cold Spring Harbor Laboratory’s Dolan DNA Learning Center. Allele
Server is a Web-based database that contains genotype data from a range of populations
around the world as well as other classrooms and teacher training workshops. It also provides
a collection of statistical analysis tools to examine the Alu insertion polymorphism at the
population level. You can either analyze your classroom data as an individual population or
compare your population with other populations in the database.
Once you enter classroom data into Allele Server, you can perform a Chi-square
analysis to compare the Alu genotype frequencies within the class population with those
predicted by the Hardy-Weinberg equation. The genotypic frequencies of the class population
can also be compared with the genotypic frequencies of another population in the database,
using the Chi-square analysis.

Using Allele Server

Note: The Dolan DNA Learning Center web site is continually updated. Some of the
following information may change.
1. On your Web browser, go to vector.cshl.org
2. Log in to Allele Server using the username and password your instructor provides.
3. Once you have logged in, follow instructions provided in the pop-up window for
using Allele Server. You may also open a new window and go to
dnalc.org/help/sad/topic_3.html to get more detailed instructions. Follow the detailed
instructions on how to populate the workspace, analyze groups, compare groups, and
query the database.
Remember that as a registered user, you may store any groups that you loaded in your
personal Allele Server database and analyze them at your convenience.

73