4.

0 Theory
The purpose of crime scene investigation is to help establish what happened and to
identify the responsible person. This is done by carefully documenting the conditions at the
crime scene and recognizing all relevant physical evidence. The ability to recognize and
properly collect physical evidence is oftentimes critical to both solving and prosecuting
violent crimes. It is no exaggeration to say that in the majority of cases, the law enforcement
officer who protects and searches a crime scene plays a critical role in determining whether
physical evidence will be used in solving or prosecuting violent crimes.
Documenting crime scene conditions can include immediately recording transient
details such as lighting (on/off), drapes (open/closed), weather, or furniture moved by
medical teams. Certain evidence such as shoeprints or gunshot residue is fragile and if not
collected immediately can easily be destroyed or lost. The scope of the investigation also
extends to considerations of arguments which might be generated in this case (suicide/self
defense) and documenting conditions which would support or refute these arguments.
Although there are common items which are frequently collected as evidence
(fingerprints, shoeprints, or bloodstains), literally any object can be physical evidence.
Anything which can be used to connect a victim to a suspect or a suspect to a victim or crime
scene is relevant physical evidence. Using the "shopping list" approach (collecting all
bloodstains, hairs, or shoeprints) will probably not result in recognizing the best evidence.
For example, collecting bloodstains under a victim's body or shoeprints from emergency
personnel will rarely answer important questions. Conversely, a single matchstick (not
usually mentioned as physical evidence) recovered on the floor near a victim's body can be
excellent physical evidence since it can be directly tied to a matchbook found in a suspect's
pocket.
Theoretically, in this experiment, we used PCR analysis to help us to find the
responsible person. Therefore, PCR is an essential prerequisite to many modern DNA
technologies and is an essential component of genetic fingerprinting. Genetic fingerprinting is
based on distinguishing individuals according to differences in their genetic material. PCR is
used to amplify specific regions in the human genome that were highly variable.

In modern genetic fingerprinting, these repetitive sequences are referred to as variable

number tandem repeats (VNTRs). Genomic regions containing these repeats are very similar
between closely related individuals, but different enough so that unrelated individuals are
highly unlikely to have the same VNTRs.
A genetic fingerprint can be directly used to match DNA found at a crime scene with
suspect DNA to ultimately secure a criminal conviction. As with traditional fingerprinting,
genetic fingerprinting requires the presence of a corresponding fingerprint from any suspect
under consideration in order to make an exact match. In other words, DNA collected at a
crime scene has to match actual suspect DNA that is already on file at the detective bureau, or
a DNA sample can be taken as required if other evidence connected to the crime scene points
towards a particular individual.
As well as using genetic fingerprinting to convict criminals, this technique also serves
as a powerful tool to prove the innocence of suspects and previous wrongly convicted
individuals. Alongside traditional fingerprint analysis, DNA fingerprinting is among the most
unambiguous methods of identifying suspects today. PCR has therefore revolutionised
forensic science and criminal investigations (Jeffreys AJ, Wilson V, Thein SL. Hypervariable
minisatellite regions in human DNA. Nature. 1985;314(6006):67-73.).
5.0 Procedure
(A) Setting up the PCR Reactions
1. 5 PCR tubes CS, A, B, C and D were labelled and placed each of them into capless
micro centrifuge tube in the foam float of ice.
2. By using a fresh aerosol barrier pipet tip for each DNA sample, 20 microliter of the
appropriate template DNA was transferred into the correctly labelled tube.
3. 20 microliter of the blue MMP (master mix + primers) was transferred into each of the
5 PCR tubes containing template DNA. The solution was mixed by pipetting up and
down. Each tube was capped after the adding of the blue MMP.
4. The capped PCR tubes were placed in their adaptors on ice.
5. The tubes were placed in the thermal cycler after we were instructed to do so. The
thermal cycler was programmed by our instructor.

(B) Electrophoresis of the PCR products

1. The gel electrophoresis equipment was set up as instructed.

2. From (A), 5 PCR tubes were obtained. The PCR tubes were placed in capless tubes
and pulse-spin in a balanced microcentrifuge for a few seconds to collect all liquid to
the bottom of the tube.
3. 10 microliter of Orange G loading dye (from tube labelled LD) was transferred into
each of the PCR tubes. The solution was pipetted up and down to mix and pulse-spin
to collect liquid in the bottom of the tube.
4. An agarose gel was placed in the electrophoresis apparatus. The wells of the agarose
gel were near the black electrode and the base of the gel was near the red electrode.
5. The electrophoresis chamber was filled with enough 1x TAE buffer to cover the gel.
6. By using a clean tip for each sample, 20 microliter of the samples were loaded into
the wells of the gel in the following order:
Lane
1
2
3
4
5
6

Sample
Allele Ladder
Crime Scene
Suspect A
Suspect B
Suspect C
Suspect D

Load Volume
20 microliter
20 microliter
20 microliter
20 microliter
20 microliter
20 microliter

7. The lid on the gel box was secured. The lid attached to the base in only one
orientation red to red and black to black. The electrical leads were connected to power
supply.
8. The power supply was turned on and the samples were electrophoresed at 100V for 30
minutes.
9. The agarose gel was then stained in Fast Blast DNA stain.