And so we come at last to the use of DNA analysis as a tool in forensic practice.

The discussion board of course

remains a great way of tailoring the content to fit the class.
Objectives
By the completion of this module the student should:
Appreciate that an individual's DNA is believed to be unique
eali!e what identical twins means at a DNA level"
Be able to review the history of DNA typing
#nderstand the uses of DNA testing
#nderstand the pitfalls of DNA testing
$ave a general overview of the technology involved
Individuality
This might seem an obvious place to begin or even to s%ip past but the concept of individuality is a surprisingly &estern and modern one at a philosophical level. Thin% bac% in history' and
in some places today' how important family vengeance was' and how guilt passed from one generation to the ne(t. The importance of individuality is thus a product' not )ust of biology' but
also of culture. But having said that we ac%nowledge that we are individuals* each of us is unique. &e %now that identical twins are genetically identical but we would also concede that they
are two individual people. +o identity and individuality may be slightly different concepts.
,n the past fingerprint evidence and other less reputable means of determining differences allowed crime investigation to focus on a single individual and confirm their pro(imity to a crime
scene. The discovery of DNA structure' and more importantly the development of readily usable technology revolutioni!ed identity matching. Both human-engendered cloned animals and
nature's e(periment .identical twins/ have DNA differences. During the life of an individual our somatic DNA is sub)ect to recombination and mutation. ,n the case of the
genes coding for immunoglobulins in B lymphocytes and T cell receptors of T lymphocytes' recombination
results in hyper-variability' allowing differences in DNA sequence even between otherwise identical twins.
&e have 01 pairs of chromosomes. ,ndividuals have 22.34 similarity* 5.34 different. 04 different and you are a
chimpan!ee. ,n forensic practice we put a huge emphasis on where we differ. 6f DNA only about 74 actually
codes for anything and the greatest variability is often in non coding regions. +o DNA testing is not about gene
transcription and phenotypic differences' it is much more centered around variable' non coding areas of
repetitive sequences that represent about a quarter of the genome.
8ingerprint matching preceded real statistics and has arguably never been robustly validated. A small number of points are compared. ,f they match then proof of identity is presumed - but
with what statistical certainty9 &ho ever as%s in court9 And what of graphology9 No statistics - so instead we rely on e(pert evidence. $ow do we %now it is believable9 $ow can its
scientific validity be tested9
&e cannot prove it' but we can have probabilities that to all intents and purposes we have established
uniqueness. :alculations depend on .i/ the type of DNA assay .ii/ the number of different tests .iii/ the degree of
relatedness .as% yourself what the effect would be if humans had multiple mono!ygotic pregnancies"/.
A Historical Perspective
,n the late ;2<5s it was reali!ed that mapping the genome could be aided by utili!ing en!ymes which cut DNA at
restricted sequence specific sites. ,ndividuals differ in where these en!ymes cut' thus giving different fragment
si!es' the restriction fragment length polymorphism .8=>/. >olymerase chain reaction .>:/ finally emerged
and made DNA technology available to any laboratory and allowed massive scaling up of effort and output.
?enome mapping and sequencing followed. By the late ;225s cloning became a reality. During this time the
forensic community had moved from loo%ing at multiple loci with large amounts of DNA being required and
+outhern blot analysis' to single locus testing and the use of >: to study tiny amounts of source DNA' that may
even be quite seriously degraded.
@(ploitation of the stability of mitochondrial DNA in historical or decomposed tissues to identify maternal identity.
Aitochondrial DNA is almost entirely inherited as a single haplotype from the mother as it is present in the
oocyte. The study of two hypervariable regions allows lineage tracing and identification of remains between
maternally unrelated sources. The study of variable regions of the B chromosome facilitated studies of paternal
lineage.
A relatively new area' with perhaps limited usefulness' is the use of typing variable DNA from immunoglobulin
coding genes in B lymphocytes' or T cell receptor re-arrangements in T lymphocytes. Antibodies are
immunoglobulin molecules and their variability reflects e(posure to bacteria and other foreign antigens. ,t is
e(pected that individuals have a unique DNA mi(ture in these genes reflecting their own personal )ourney in a
hostile environment. @pigenetic variation. The effect of the stamp embossed upon the same underlying genetic
sequence mar%ing it as being maternally and paternally derived appears to have a significant phenotypic effect
in some situations. Bou may want to delve further in to this area by loo%ing in to the genetics of Angelman and
>rader-&illi syndromes.
+ome Technical and 6ther >itfalls
The best place to learn the practicalities of DNA testing is in an operational laboratory with bac%up perhaps in an
academic laboratory speciali!ing in teaching quality assurance and control. We will mention some in no
particular order and encourage you to extend the list and discussion on the discussion board.
1) Contamination
As in every e(ample where you are see%ing to gather evidence' the sampling procedures
used and the degree of technical s%ill' verified by good quality assurance protocols' must be
of the highest standards. :ontamination can occur at many stages in the process from mi(ing
samples' to carry over from one sample to another when loading a gel. This is why standard
operating procedures' good practice' audit and quality assurance schemes are all essential
for a truly scientific laboratory. A further important point to emphasi!e is that collection in the
field if improperly performed ruins everything that follows' no matter how good the science is.
>rotocols must start at the scene of a crime and evidence gathering. +traying from this goal
discredits the laboratory and may let the guilty escape or convict the innocent.
Degradation
&arm humid conditions' bacterial contamination from soil' gastric contents' e(posure to the air' decomposition'
fire and chemicals may all cause DNA to degrade. 8or the classical approach of 8=> analysis large amounts of
DNA in good condition are required. Any of the factors above may degrade the DNA to small' unusable
fragments. >: can deal more readily with small fragments' and mitochondrial DNA may survive longer in a
useful form. Tooth pulp may be a useful repository for DNA if all else fails. After collection of a sample it is
important to stop further degradation as quic%ly as possible. Degraded DNA results in a failed test* it does not
result in a changed DNA profile. ,f a >: assay gives a positive result or a negative one .that is no band at all/
there is a danger of a failed assay being mista%en for a negative result. Calidation of DNA quality is always a
useful chec% as well as optimal design of >: strategies.
Mixtures
,n a rape the ma)ority of DNA collected may be from the victim' especially if there is bleeding. ,f the assailant has
had a vasectomy or is otherwise a!ospermic .too few sperm/ there may be very little of the assailant's DNA to
deal with. Approaches developed to analy!e such samples must ta%e in to consideration that the assailant's DNA
may be a minority of the sample and care must be ta%en to ma(imi!e its usefulness. 8ind out what techniques
can be employed to try to overcome such difficulties and share your findings with your colleagues on the
discussion board. Bou might wish to speculate on the discussion board whether there are other problems
associated with analysis of very small amounts of DNA from sperm' which originate as a result of meiotic division
and are' therefore' haploid.
Approaches have been developed to try to deduce which alleles in a mi(ed sample come from the same source
on the basis of the strength of signal the different alleles produce when they are detected. The theory being that
if you have four alleles at a locus' two of which give a very strong signal of equivalent strength and two of which
give a much wea%er signal of equivalent strength the two stronger alleles came from one individual and the two
wea%er alleles from another individual. This may seem plausible if we surmise that the DNA from one of the
contributors was in e(cess of the other in the sample analy!ed' but what other alternatives might have given rise
to this result9 :onsider the scenario when we have 3 pea%s - how many different individuals might have
contributed to the sample9 Thin% about all the scenarios where we have individuals who are hetero!ygous for
alleles' homo!ygous for alleles and the situation where individuals share alleles in common. emember we may
not always have a distinction in the pea% height that might provide a clue to the allele allocation. Ai(ture analysis
is comple( and should not be considered definitive.
"Innocent" presence of DNA
DNA is fairly stable given reasonable environmental conditions and so its presence in space cannot be
linked to its presence in time - that is the time of a crime. The ability to detect increasingly smaller
traces of DNA by techniques such as LCN Lo! Copy Number" DNA analysis should ring alarm bells
!ith the competent scientist. #e!are the temptation to attach greater significance to the fact that a
profile might be detected. The presence of my DNA does not even confirm my presence at the scene.
Quait! assurance
6ne of the other courses offered through the #niversity of 8lorida stresses the importance of quality assurance
and control. DNon-academicE laboratory has much more stringent standards and procedures. The forensic
laboratory cannot afford a false positive later corrected' or a false negative' and there is no scope for as%ing for a
better sample because the free!er bro%e down.
"rosecutor#s faac!
The level of proof we require will vary. ,dentification of lineage in an anthropological survey is less stringent than
the identification of an alleged rapist. ,n practice a number of assays are performed that lead to a degree of
unli%eliness that anyone other than the suspect is the origin of the DNA. But it is only a degree of probability' a
confidence limit. ,f , perform a DNA profile and find , calculate that the probability of a DNA match with someone
other than the suspect is ; in ;5F what am , saying9
=et's suppose there are not reasons locally' such as inbreeding and an identical twin' to complicate matters' we
can say confidently that we cannot e(clude the suspect with a probability of being right 222'222 out of ;'555'555
times. 6f course a further set of polymorphic mar%ers may )ust prove that one time in a million and we can go on
and e(clude involvement. ,t is apparent that the fewer tests we use .the fewer polymorphic mar%ers we loo% at/
the more li%ely we are to fail to e(clude an innocent party as a possible source of the DNA. The prosecutor's
fallacy is to transpose the unli%eliness of being able to e(clude guilt .; in ;5F / as being the same as the
probability of guilt and there is' therefore' 222'222 chances in ;'555'555 that the suspect is guilty as charged"
&e had better mention the defender's fallacy as well' if there is a one in a million chance of this not being the
suspect's DNA then in a country with a population of 155 million' 022 other people will match this DNA profile.
Therefore' the accused has a 022 in 155 chance of being innocent" :onfused9 , hope not"
DNA @(traction and >olymerase :hain reaction .>:/
6ptimal quality' fresh DNA material is not usually available in forensic investigations and so e(traction is a critical
phase of the DNA profiling procedure to ensure there is ma(imal return of quality' quantity and freedom from
contaminant chemical impurities that inhibit >: reactions. Details on e(traction and other protocols can be
obtained and updated from websites we have previously e(plored for those who wish to loo% in to the practice.
>olymerase chain reaction .>:/ is an invaluable technique' because it can be used .i/ on small samples .ii/
small fragments' for e(ample those obtained from degraded DNA .iii/ much of the process can be automated .iv/
it is rapid .v/ it does not require use of radioactive probes. The %ey is to design oligonuleotide primers' which we
commonly refer to as primers' that correspond to genomic nuclear or mitochondrial DNA. #nder appropriate
conditions these anneal to their complementary sequence in the sample DNA' which has ta%en up the single
stranded form. The addition of a polymerase en!yme produces a new strand of DNA at the site of interest. The
really neat bit in >: is the en!yme is thermostable. &e can heat the sample to separate the new strands from
the original template in order to get the single stranded form again and on cooling' the strands recombine' but
because there is still lots of primer' these will anneal to their complementary sequence and we can again
generate new strands as the en!yme has not been denatured by heat. :ycle after cycle of this process results in
an e(ponential increase in the target sequence quite specifically. $ence' the name polymerase chain reaction' or
>:.
Restriction Fragment Length Polymorphism (RFLP)
estriction =ength 8ragment >olymorphism .8=>/ technique - original method of DNA analysis applied to
forensic casewor% bac% in the ;2G5s* dubbed EDNA fingerprintingE. &ith 8=> analysis more DNA is
required than for the techniques currently employed and the DNA must have a larger fragment si!e. DNA
aliquots are digested with restriction en!ymes that cut only at specific %nown sites in the DNA strand. When
DNA rom dierent individuals is digested! polymorphisms result in dierent si"es ater cutting #ith the
restriction en"yme$ These can be separated by electrophoresis on a gel according to si!e. The gel is made of
polyacrylamide which acts li%e a sieve with pores which form in the gel as it EsetsE or crosslin%s. &hen a
potential difference is applied across the gel the negatively charged DNA molecules migrate through the gel from
the cathode .negative electrode/ to the anode .positive electrode/. The smaller the DNA fragment the easier it is
for it to EwiggleEthrough the gel. =arger fragments are retained in the gel for longer an travel through the gel at a
rate that is inversely proportional to their length. After separation the entire genomic DNA' comprising different
si!ed fragments' is present in the gel in the form of a smear. Then we must identify and highlight the variable
regions we are interested in and then visuali!e them to record the presence of bands which will be unique to the
individual if sufficient different sites are studied. Cisuali!ation again relies on the property of DNA when
denatured to lose its double strand conformation on heating but regain it on cooling. ,nstead of interposing a
primer on cooling the tric% for 8=> analysis is to have a short probe made from the target sequence' labeled
with radioactivity or some other mar%er. This binds to the denatured single stranded DNA fragments and can be
detected by detecting the radioactivity or fluorescence. Because the probe binds the digested genomic DNA
wherever it migrated in the gel the si!e of visuali!ed fragments can be measured and different samples
compared.
+o &hat +trategy +hould &e #se9 #p to ;3 sites of e(tremely variable DNA can be studied by 8=>
analysis. The limitations are time! DNA %uantity and %uality but the results can be e(tremely accurate and
thorough' allowing really impressive statistical significance. 6riginally it relied upon a probe that bound to a
number of different loci. Newer applications have tended towards single locus probes that increase the
stringency of hybridi!ation reactions. #se of four single locus probes can give very good DNA profiles that are
technically repeatable' but of course the downside is that the power of discrimination .that is the ability to
e(clude samples as being the same' or to confirm uniqueness/ is reduced.
$%& "rofiing
The use of short tandem repeats .+T/ as a target e(ploits using mar%ers which are highly variable. +Ts are
preferred to larger polymorphisms .variable number tandem repeats CNTs/ because of the attractiveness of
being able to automate +T analysis and use >: on degraded DNA. +Ts are based on loci with
tetranucleotide repeats with nigh on 25 different alleles at some of the loci tested' for e(ample the D0;+;;
locus
;
. The different alleles differ in the number of times the particular tetranucleotide being analy!ed is
repeated at the locus' which may vary from a few to do!ens of times. Because of the small si!e of the +T loci
and e(treme variability >: analysis is an ideal approach and indeed several different loci may be studied in the
same >: reaction. ,f fluorescent labeled nucleotides are used the results can be read by a DNA sequencer'
allowing greater throughput and standardi!ation.
Traditional DNA sequencers utili!ed vertical polyacrylamide slab gels to separate the >: products on the basis
of their si!e. $owever' in recent years there has been a shift to separate the fragments by capillary
electrophoresis. ather than having a large slab gel of polyacrylamide' capillary electrophoresis uses fine
capillaries of 35-;55Hm diameter which are pac%ed with a viscous polymer through which the DNA fragments
migrate. The polymer serves the same purpose as the polyacrylamide EsieveE but the capillary electrophoresis
system has the advantage in being able to separate a sample in minutes rather than hours' because higher
voltages can be used in the separation. Ainute amounts of sample are needed for the analysis' which ma%es it
preferable when samples are in short supply and increases the possibility for reanaly!ing samples if need be.
The system can be fully automated .in)ecting the sample' separating the fragments and fragment detection/ and
there is no ris% of cross-contamination between lanes of the gel or a need to trac% lanes. A drawbac% of the
capillary electrophoresis system is the throughput rate because samples have to be analy!ed sequentially
through the capillary tube' rather than side by side as on a conventional polyacrylamide gel. $owever' capillary
arrays have now been developed such as the Applied Biosystems 1;15(l ?enetic Analy!er' ;F capillary system
shown which has ;F capillary tubes.
,f four or five +T loci are e(amined this gives a high level of discrimination between samples and individuals'
but the number of loci studied can be increased depending on bac%ground variability .for e(ample' related to
ethnicity/ and the importance of e(clusion in the case being e(amined. The more loci that are used to generate a
DNA profile the less li%ely it is to get an adventitious match.
The 8B, uses ;1 +T loci to type DNA of samples entered into the #+ national database' which is %nown as
ND,+ .National DNA ,nde( +ystem/. The software for management of the database and profile comparisons is
called :6D,+ .:ombined DNA ,nde( +ystem/' and is used for ND,+ as well as databases at #.+. state .+D,+/
and local .=D,+/ crime lab levels' and also in some non-#.+. countries. :ommercially available %its are used that
include the core ;1 +T loci amongst a total of ;3 or more loci. The #I National DNA Database
J
stores profiles
produced from commercially available %its for e(ample %nown as +?A >lusK .+econd ?eneration Aultiple(
>lus/ which loo%s at ;5 +T loci. The li%elihood of obtaining a match from two unrelated individuals by chance
when comparing two full +?A >lusK profiles is presented in :ourt as being in the order of ; in a billion.