TITLE: Development of an Alu-based DNA fingerprinting system suitable for general

applications in teaching environment.

Author: Sarah Jane Welford (Undergraduate student at Chester University).
Published in Origin, 2004, pp57-66.

Adapted for education purposes by Dr. Maggy FOSTIER.

This aims to give you an example (i.e. this is not a model to be replicated verbatim) of how a lab
report could to be presented. NB: This
- The introduction section sets the scene, puts the experiment into context, and clearly states
the aims of the study. You will notice that facts/statements are all backed up by evidence or
explanations (like in an essay).
- The methods and materials section gives enough information to allow another researcher to
repeat your experiment. For a computer-based experiment, you should outline your
experimental design here.
- The results section only contains one figure illustrating how to present a figure. It also
shows how to describe results in the text. You will notice that the purpose of each step is
always introduced first to guide the reader. Small conclusions can be drawn to make the
logical link with the next experiment, but the full analysis is to be done in the discussion.
- The discussion section reviews the results in light of the aims of the study. Ideally it needs
to refer to other sources of information to put your analysis into context. It can address
both experimental or theoretical issues. You will notice that facts/statements are all backed
up by evidence or explanations (like in an essay). NB: here the discussion is of an
advanced level.
- Note that references (from textbooks, websites and research papers) were included in the
introduction and the discussion.
-

INTRODUCTION:
In the past five years, short tandem repeat (STR) polymorphism analysis (see introduction
to STRs in the paternity test simulation) have been developed into one of the most rapid,
efficient, and precise methods for human identification in forensic applications (reviewed
in Inman and Rudin, 2001). The amplification by polymerase chain reaction (PCR) of these
STR loci has progressed from single locus PCR reactions to multiplex PCR reactions,
amplifying as many as sixteen loci in one single reaction tube. In parallel, the detection of
STR loci has advanced from manual radioactive and silver stain detection to automated
detection using fluorescent instrumentation.
Whilst STR analysis has the discrimination power required for forensic applications, the
necessity of specialised equipment to resolve and detect STR alleles makes the technique
less suitable for more general applications, such as for educational purposes. Given the
popular interest for forensic science in school and higher education, there is therefore a
considerable need for DNA fingerprinting systems that sacrifice some power of
discrimination for ease of manipulation and applicability in less specialised laboratories.
One system potentially capable of fulfilling the criteria required for general teaching
purposes is the use of the Alu elements polymorphisms (Novick et al., 1993). Alu elements
appear to be redundant genetic sequences, probably of viral origin, which have been
integrated by transposition into many sites within the human genome during evolution
(Batzer et al., 1996; Mighell et al., 1997). Statistical examination of Alu polymorphisms at
various loci suggests that the alleles are now fixed in their respective chromosomal
locations resulting in genotypes that appear to be in equilibrium within populations and
races (Sajantila, 1998; Nasidze et al., 2001; Watkins et al., 2003). This very high stability
once inserted, combined with their widespread distribution throughout the human
genome, make Alu elements useful genetic markers for human identification (Novick et
al., 1993).
Technically, Alu polymorphism analysis is easy to establish in the teaching laboratories
for two reasons. Firstly, Alu elements are approximately 300bp long and therefore the
presence or absence of the Alu insert (+ allele or allele respectively) can be easily
determined by PCR followed by agarose gel electrophoresis to resolve the alleles.
Secondly, as flanking DNA sequences are known for many Alu insertion sites, primers can
be easily designed to allow the specific detection of the Alu polymorphism at these sites.
Although the possible genotypes for any single Alu loci are limited to +/+, -/-, or +/-, it
can be speculated that the combined use of several independently assorting Alu loci could
provide an acceptable power of discrimination for general use in science education.
The present study aimed to design a multiplex DNA fingerprinting system (where several
PCR are performed in a single tube) suitable for educational purposes based on analysis of
Alu polymorphisms. Four Alu sites (ACE, TPA-25, PV-92 and 182) have been considered
in this study. These were chosen because they are located on four separate chromosomes
and because it was possible to design 4 sets of primers with the same annealing

temperature that will produce a range of PCR fragment size that can be resolved on a
single agarose gel.
MATERIALS AND METHODS.
DNA extraction from cheek cells.
Twenty people volunteered a DNA sample for the study. Each subject was given a sterile
swab and asked to rub against the inside of their mouth for 30 sec each side. The swabs
were then vigorously swirled in an eppendorf tube with 1.5 ml of 0.9% NaCl solution to
facilitate cheek cell removal. Cells were collected by centrifugation at 10000 rpm for 10
min, leaving a distinct cell pellet. The supernatant was carefully discarded and 500 l of a
10% Chelex beads (from Sigma) suspension (in sterile water) was added to the pellet. The
pellet was resuspended in the bead solution and the tube was incubated at 100C for 10
min and then cooled on ice for a minimum of 2 min. Each reaction was then subjected to a
further centrifugation step (10 000 rpm for 1 min) to pellet the Chelex/cell debris. The
supernatant containing the genomic DNA was transferred into a sterile eppendorf tube
and stored on ice or at -20C.
PCR reaction:
Genomic DNA (10 l) was transferred into a sterile PCR tube to which was added 15 l of
BioradTM PCR mix 2X (containing buffer, enzymes, dNTPs), and 5 l of primer master mix
(final concentration of each primer: 40 ng/ l). The tubes were then placed into the
thermocycler (TECHNE 3100) and the following PCR conditions were applied: 94C for 1
min (initial denaturation), 32 cycles of 94C for 30 sec (denaturation), 55C for 30 sec
(primer annealing), 72C for 1 min (extension), then 72C for 5 min (final extension) and
finally 4C soak. The PCR products were then stored on ice or at -20C.
Agarose gel electrophoresis.
The DNA fragments generated by the PCR reactions were resolved by electrophoresis on a
2% agarose gel at 90 V (constant voltage) for 120 min. 10 l of each PCR product was
loaded with 3 l of loading buffer per well and 1 ng of the BioradTM 100 bp DNA ladder
was run on each gel for size comparison.

RESULTS:
Analysis of the Alu polymorphism at each locus separately.
Before testing if the four Alu loci (ACE, TPA-25, PV-92 and 182) could be used together
within a multiplex system, the Alu polymorphism at each site had to be characterised
individually. This experiment was performed to test if the PCR conditions were adequate
and to establish the Alu DNA profiles of each individual (in four separate PCR reactions),
which were to be compared to the results obtained with the multiplex system (in one
single PCR reaction).
Twenty unrelated persons volunteered a DNA sample for the study. For each DNA
sample, 4 different PCR reactions were undertaken, each using the set of primers
associated with one Alu locus. These experiments revealed the size of the fragments
generated for each Alu locus depending on the presence (+ allele) or absence (-allele) of
the Alu insert (data not shown). The TPA25- and TPA+ alleles generate fragments of
100bp and 400bp respectively. The ACE- and ACE+ alleles generate fragments of 190bp
and 490bp respectively. The 182- and 182+ alleles generate fragments of 287bp and 587bp
respectively. The PV92- and PV92+ alleles generate fragments of 360bp and 660bp
respectively. These sizes match the expected sizes from the sequence data showing that
the PCR conditions are adequate for each Alu locus.
Analysis of the Alu polymorphism at two loci at a time.
Before testing the four sets of primers in a single PCR, each set of primers was first tested
in pairs to investigate if a pair of primers may be dominant over another. Six PCR were
set up on 3 volunteers DNA with the primer sets of TPA25 and ACE ; TPA25 and 182;
TPA25 and PV92; ACE and 182; ACE and PV92; 182 and PV92, respectively. For each
primer combination, the fragments generated by each locus were clearly evident on the
gel (data not shown) and of similar intensity, suggesting that no primer pair dominated
the reaction.
Production of human DNA profiles using the multiplex Alu systems.
This time, the DNA of each of the 20 volunteers was subjected to one PCR only, containing
the four sets of primers. In each case, amplification of alleles from all Alu loci was
achieved again demonstrating amplification from each of the primer sets in the reaction
(the gel of the subjects 1 to 7 is shown in Figure 1). For each subject, results obtained from
that single PCR were compared with the results obtained by the PCR at each locus. The
comparison showed that they matched, proving that the multiplex system was functional.
Interestingly, when considering the four loci, none of the 20 subjects showed an identical
DNA profile, illustrating that the multiplex Alu system has a fair degree of discrimination
for a small group.

Figure1. Example of the results obtained with the multiplex Alu system
(ACE, TPA-25, PV-92 and 182).
The genomic DNA of 7 people was amplified in a multiplex PCR reaction
(with four sets of primers) and the PCR fragments were resolved on a 2%
agarose gel. Lane 1 (on the far left) contains 1ng of the 100 bp DNA
ladder, lane 2-8 contains 10 l of each PCR reaction. The person on lane 8
(on the far right) is heterozygous for the four Alu loci, i.e. has 8 different
alleles. The size and genotype of each allele is indicated on the right of
lane 8; + stands for the presence of the Alu insert, - stands for the
absence of the insert.

DISCUSSION.
To use a DNA typing system in the educational laboratory environment, the method for
DNA extraction has to present the minimum of hazard to the user and be robust enough
to produce quality extracts even when used by non-specialised personnel, e.g. school
children or 1st year undergraduate students. In addition, it is important that the method is
non-invasive to reduce the potential risk of either injury or infection. The Chelex-based
methodology of DNA extraction from buccal cells used in this experiment is cheap, rapid,
simple and non invasive. It also proved to be effective for the purpose of the experiment
for two reasons. Firstly, the yield from a single cheek cells sample was found to be highly
reproducible and sufficient for multiple PCR. This allowed the procedure to move directly
to the PCR step without having to check that there was enough DNA template, thereby
removing the need of spectrophotometric analysis to determine the DNA sample
concentration. Secondly, the quality of the DNA samples were good enough for a
downstream application as sensitive as PCR, suggesting that the concentration of potential
PCR inhibitors in the final extract was negligible. In all cases, 10 l of DNA template
aliquots were found to produce highly readable DNA fingerprints following
amplification.
Visualisation of DNA fragments by agarose gel electrophoresis can easily and rapidly be
achieved with a minimum of hazard (the users have to wear gloves and labcoats) by
including ethidium bromide into the gel. This is a molecule that intercalates between DNA
base pairs and is highly fluorescent under UV light, thus revealing the DNA fragments as
bands in the gel. For schools that may lack the necessary equipment, 100% safe stains have
now been developed; the only inconvenience is that they require a long time period for
detection (BIORADTM safe stain).
The four Alu primer sets chosen for the multiplex PCR system were designed following
two constraints: they needed to have very similar annealing temperatures to function in a
single PCR reaction and they needed to generate DNA fragments of significantly different
sizes so that they could easily be separated by agarose gel electrophoresis. These
constraints can be the source of two known problems with PCR multiplex systems.
Firstly, PCR where variants in amplicons (PCR products) size are possible may favour
increased amplification of smaller amplicons. In DNA typing, this has been shown to lead
to a phenomenon known as allelic dropout where some alleles, because of their size or
preference for certain PCR conditions, may be over or under represented following
amplification (Miller et al., 2002; Dreesen et al., 1996). Secondly, a known problem when
multiplex PCR is used appears to be the failure of some alleles to amplify when
competition from other primers occur in the reaction.
In our study, preliminary experiments were performed where all primer sets were tested
individually and then in all the possible pairs combinations. In all cases, amplifications of
alleles for each locus were discernible in the dual reactions and the presence of double
heterozygotes confirmed that no allelic dropout was occurring. This showed that our
primer sets were not in competition with each others and that our amplicons sizes span
(from 100pb to 660pb) was not source of a problem. When the multiplex system was

tested, the 8 alleles of a quadruple heterozygote volunteer were equally amplified,

confirming that the system was effective at amplifying all possible alleles reliably.
The four Alu loci chosen for our multiplex have already been studied and it was shown
that there alleles are in at the Hardy Weinberg equilibrium within populations and races
(Sajantila, 1998; Nasidze et al., 2001; Watkins et al., 2003). This feature was essential in our
choice of Alu polymorphisms as markers of individual genetic variation for our multiplex
system. It means that we can assume that the allelic distribution of the Alu inserts is
uniform within the population and therefore that the probability of two DNA fingerprints
chosen at random matching using the Alu multiplex as in the present study is 34 (1 in 84).
Our study revealed that our 20 volunteers all had unique fingerprint which is in
concordance with a prediction based on the above analysis. It should be noted however,
that our 20 volunteers were all Caucasians and therefore that the system needs to be
further tested on a larger population with a diverse racial mix to establish more precisely
its power of discrimination.
To conclude, our study has demonstrated that the multiplex Alu system (ACE, TPA-25,
PV-92 and 182) is suitable for educational purposes, as it is inexpensive, rapid, easy to use,
and provides reproducible results. The systems power of discrimination needs to be
further tested on different populations, but our study suggests that the discriminatory
power is high enough for small sample populations such as a typical class size. Finally, it
is worth noticing that this system can also be used to introduce population genetics
concepts in the classroom and notably test if the class is at Hardy Weinberg equilibrium
for each of the Alu alleles typed.

REFERENCES:
Batzer MA, Arcot SS, Phinney JW, Alegria-Hartman M, Kass DH, Milligan SM, Kimpton C, Gill
P, Hochmeister M, Ioannou PA, Herrera RJ, Boudreau DA, Scheer WD, Keats BJ, Deininger PL,
Stoneking M. (1996). Genetic variation of recent Alu insertions in human populations.
Journal of Molecular Evolution 42(1): pp 22-9.
Dreesen JC, Bras M, Coonen E, Dumoulin JC, Evers JL, Geraedts JP. (1996). Allelic dropout
caused by allele-specific amplification failure in single-cell PCR of the cystic fibrosis
delta F508 deletion. Journal of Assisted Reproductive Genetics 13(2): pp 112-4.
Inman K and Rudin N (2001), An Introduction to Forensic DNA, 2nd edition, CRC Press
Miller CR, Joyce P, Waits LP. (2002). Assessing allelic dropout and genotype reliability
using maximum likelihood. Genetics 160(1): pp 357-66.
Mighell AJ, Markham AF, Robinson PA. (1997). Alu sequences. FEBS Letters 417(1):1-5.
Nasidze I, Risch GM, Robichaux M, Sherry ST, Batzer MA, Stoneking M. (2001). Alu insertion
polymorphisms and the genetic structure of human populations from the Caucasus.
European Journal of Human Genetics. 9(4): pp 267-72.
Novick GE, Gonzalez T, Garrison J, Novick CC, Batzer MA, Deininger PL, Herrera RJ. (1993).
The use of polymorphic Alu insertions in human DNA fingerprinting. EXS 67:283-91.
Sajantila A. (1998). A World Wide Survey on Human Specific Alu Insertion
Polymorphisms. http://www.promega.com/geneticidproc/eusymp2proc/01.pdf, last
accessed 2006.
Watkins WS, Rogers AR, Ostler CT, Wooding S, Bamshad MJ, Brassington AM, Carroll ML,
Nguyen SV, Walker JA, Prasad BV, Reddy PG, Das PK, Batzer MA, Jorde LB. (2003). Genetic
variation among world populations: inferences from 100 Alu insertion polymorphisms.
Genome Research: 13(7): pp 1607-18.