MULUNGUSHI UNIVERSITY

STUDENT NAME: KHANYISO KALYATA

STUDENT NUMBER: 202003527
PROGRAM: LABORATORY TECHNOLOGY CHEMISTRY MAJOR
COURSE CODE: SLT 371
LAB ASSIGNMENT
Polymerase Chain Reaction in DNA Fingerprinting and
Forensic Analysis

Polymerase chain reaction, or PCR, is a technique to make many

copies of a specific DNA region in vitro (in a test tube rather than an
organism). The polymerase chain reaction (PCR) is a popular method to
copy DNA in vitro. Its invention revolutionized fields ranging from clinical
medicine to anthropology, molecular biology, and forensic biology. The
method employs one of many available heat-stable DNA polymerases in
a reaction that is repeated many times in situ. The DNA polymerase
reads a template DNA strand and using the components of the reaction
mix, catalyzes the addition of free 2'-deoxynucleotide triphosphate
nitrogenous bases to short segment of DNA that forms a complement
with the template via Watson-Crick base pairing. This short segment of
DNA is referred to as a PCR primer and it is essential to the success of
the reaction.

Using PCR, a DNA sequence can be amplified millions or billions of

times, producing enough DNA copies to be analyzed using other
techniques. For instance, the DNA may be visualized by gel
electrophoresis, sent for sequencing, or digested with restriction
enzymes and cloned into a plasmid.

PCR is used in many research labs, and it also has practical applications
in forensics, genetic testing, and diagnostics. For instance, PCR is used
to amplify genes associated with genetic disorders from the DNA of
patients (or from fetal DNA, in the case of prenatal testing). PCR can
also be used to test for a bacterium or DNA virus in a patient's body: if
the pathogen is present, it may be possible to amplify regions of its DNA
from a blood or tissue sample.
PCR can be used as a tool in genetic fingerprinting. This technology can
identify any one person from millions of others. For example, tiny
samples of DNA isolated from a crime scene can be compared with DNA
from suspects, or compared with a DNA database. Such procedures can
identify or rule out suspects during a police investigation. PCR-based
DNA fingerprinting can also be used in parental testing in which an
individual is compared with their close relatives and the actual biological
father of a child can be confirmed or ruled out. DNA testing can also
confirm the biological parents of an adopted child.
Because PCR amplifies the regions of DNA that it targets, PCR can be
used to analyze extremely small amounts of sample. This is often critical
for forensic analysis, when only a trace amount of DNA is available as
evidence. PCR may also be used in the analysis of ancient DNA that is
tens of thousands of years old.
STAGES IN PCR REACTION

Denaturation: This step is the first regular cycling event and consists of
heating the reaction chamber to 94–98 °C (201–208 °F) for 20–30
seconds. This causes DNA melting, or denaturation, of the double-
stranded DNA template by breaking the hydrogen bonds between
complementary bases, yielding two single-stranded DNA molecules.

Annealing: In the next step, the reaction temperature is lowered to 50–

65 °C (122–149 °F) for 20–40 seconds, allowing annealing of the
primers to each of the single-stranded DNA templates. Two different
primers are typically included in the reaction mixture: one for each of the
two single-stranded complements containing the target region. The
primers are single-stranded sequences themselves, but are much
shorter than the length of the target region, complementing only very
short sequences at the 3' end of each strand.

Extension/elongation: The temperature at this step depends on the DNA

polymerase used; the optimum activity temperature for the thermostable
DNA polymerase of Taq polymerase is approximately 75–80 °C (167–
176 °F),[14][15] though a temperature of 72 °C (162 °F) is commonly used
with this enzyme. In this step, the DNA polymerase synthesizes a new
DNA strand complementary to the DNA template strand by adding free
dNTPs from the reaction mixture that is complementary to the template
in the 5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs
with the 3'-hydroxy group at the end of the nascent (elongating) DNA
strand. The precise time required for elongation depends both on the
DNA polymerase used and on the length of the DNA target region to
amplify.

The development of PCR-based genetic (or DNA) fingerprinting

protocols has seen widespread application in forensics:

DNA samples are often taken at crime scenes and analysed by PCR.

In its most discriminating form, genetic fingerprinting can uniquely

discriminate any one person from the entire population of the world.
Minute samples of DNA can be isolated from a crime scene,
and compared to that from suspects, or from a DNA database of
earlier evidence or convicts. Simpler versions of these tests are often
used to rapidly rule out suspects during a criminal investigation.
Evidence from decades-old crimes can be tested, confirming
or exonerating the people originally convicted.

 Forensic DNA typing has been an effective way of identifying or

exonerating criminal suspects due to analysis of evidence discovered
at a crime scene. The human genome has many repetitive regions
that can be found within gene sequences or in non-coding regions of
the genome. Specifically, up to 40% of human DNA is repetitive.
[5]
 There are two distinct categories for these repetitive, non-coding
regions in the genome. The first category is called variable number
tandem repeats (VNTR), which are 10–100 base pairs long and the
second category is called short tandem repeats (STR) and these
consist of repeated 2–10 base pair sections. PCR is used to amplify
 several well-known VNTRs and STRs using primers that flank each of
the repetitive regions. The sizes of the fragments obtained from any
individual for each of the STRs will indicate which alleles are present.
By analyzing several STRs for an individual, a set of alleles for each
person will be found that statistically is likely to be unique.
[5]
 Researchers have identified the complete sequence of the human
genome. This sequence can be easily accessed through the NCBI
website and is used in many real-life applications. For example, the
FBI has compiled a set of DNA marker sites used for identification,
and these are called the Combined DNA Index System (CODIS) DNA
database.[5] Using this database enables statistical analysis to be
used to determine the probability that a DNA sample will match. PCR
is a very powerful and significant analytical tool to use for forensic
DNA typing because researchers only need a very small amount of
the target DNA to be used for analysis. For example, a single human
hair with attached hair follicle has enough DNA to conduct the
analysis. Similarly, a few sperm, skin samples from under the
fingernails, or a small amount of blood can provide enough DNA for
conclusive analysis.[5]
 Less discriminating forms of DNA fingerprinting can help in DNA
paternity testing, where an individual is matched with their close
relatives. DNA from unidentified human remains can be tested, and
compared with that from possible parents, siblings, or children.
Similar testing can be used to confirm the biological parents of an
adopted (or kidnapped) child. The actual biological father of a
newborn can also be confirmed (or ruled out).
 The PCR AMGX/AMGY design facilitate in amplifying DNA
sequences from a very minuscule amount of genome. However it can
also be used for real-time sex determination from forensic bone
samples. This provides a powerful and effective way to determine
gender in forensic cases and ancient specimens.
Gel Electrophoresis in DNA Finger Printing and Forensics

Electrophoresis is a process that enables the sorting of molecules based

on size. Using an electric field, molecules (such as DNA) can be made to
move through a gel made of agarose or polyacrylamide. The electric
field consists of a negative charge at one end which pushes the
molecules through the gel, and a positive charge at the other end that
pulls the molecules through the gel. The molecules being sorted are
dispensed into a well in the gel material. The gel is placed in an
electrophoresis chamber, which is then connected to a power source.
When the electric field is applied, the larger molecules move more slowly
through the gel while the smaller molecules move faster. The different
sized molecules form distinct bands on the gel.
The term "gel" in this instance refers to the matrix used to contain, then
separate the target molecules. In most cases, the gel is a crosslinked
polymer whose composition and porosity are chosen based on the
specific weight and composition of the target to be analyzed. When
separating proteins or small nucleic acids (DNA, RNA,
or oligonucleotides) the gel is usually composed of different
concentrations of acrylamide and a cross-linker, producing different
sized mesh networks of polyacrylamide. When separating larger nucleic
acids (greater than a few hundred bases), the preferred matrix is purified
agarose. In both cases, the gel forms a solid, yet porous matrix.
Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be
handled using appropriate safety precautions to avoid poisoning.
Agarose is composed of long unbranched chains of uncharged
carbohydrates without cross-links resulting in a gel with large pores
allowing for the separation of macromolecules and macromolecular
complexes.
Electrophoresis refers to the electromotive force (EMF) that is used to
move the molecules through the gel matrix. By placing the molecules in
wells in the gel and applying an electric field, the molecules will move
through the matrix at different rates, determined largely by their mass
when the charge-to-mass ratio (Z) of all species is uniform. However,
when charges are not all uniform the electrical field generated by the
electrophoresis procedure will cause the molecules to migrate
differentially according to charge. Species that are net positively charged
will migrate towards the cathode which is negatively charged (because
this is an electrolytic rather than galvanic cell), whereas species that are
net negatively charged will migrate towards the positively charged
anode. Mass remains a factor in the speed with which these non-
uniformly charged molecules migrate through the matrix toward their
respective electrodes.
If several samples have been loaded into adjacent wells in the gel, they
will run parallel in individual lanes. Depending on the number of different
molecules, each lane shows the separation of the components from the
original mixture as one or more distinct bands, one band per component.
Incomplete separation of the components can lead to overlapping
bands, or indistinguishable smears representing multiple unresolved
components.[citation needed] Bands in different lanes that end up at the same
distance from the top contain molecules that passed through the gel at
the same speed, which usually means they are approximately the same
size. There are molecular weight size markers available that contain a
mixture of molecules of known sizes. If such a marker was run on one
lane in the gel parallel to the unknown samples, the bands observed can
be compared to those of the unknown to determine their size. The
distance a band travels is approximately inversely proportional to the
logarithm of the size of the molecule (alternatively, this can be stated as
the distance traveled is inversely proportional to the log of samples's
molecular weight).
There are limits to electrophoretic techniques. Since passing a current
through a gel causes heating, gels may melt during electrophoresis.
Electrophoresis is performed in buffer solutions to reduce pH changes
due to the electric field, which is important because the charge of DNA
and RNA depends on pH, but running for too long can exhaust the
buffering capacity of the solution. There are also limitations in
determining the molecular weight by SDS-PAGE, especially when trying
to find the MW of an unknown protein. Certain biological variables are
difficult or impossible to minimize and can affect electrophoretic
migration. Such factors include protein structure, post-translational
modifications, and amino acid composition. For example, tropomyosin is
an acidic protein that migrates abnormally on SDS-PAGE gels. This is
because the acidic residues are repelled by the negatively charged SDS,
leading to an inaccurate mass-to-charge ratio and migration. [8] Further,
different preparations of genetic material may not migrate consistently
with each other, for morphological or other reasons.
The types of gel most typically used are agarose and polyacrylamide
gels. Each type of gel is well-suited to different types and sizes of the
analyte. Polyacrylamide gels are usually used for proteins and have very
high resolving power for small fragments of DNA (5-500 bp). Agarose
gels, on the other hand, have lower resolving power for DNA but have a
greater range of separation, and are therefore used for DNA fragments
of usually 50–20,000 bp in size, but the resolution of over 6 Mb is
possible with pulsed field gel electrophoresis (PFGE).[9] Polyacrylamide
gels are run in a vertical configuration while agarose gels are typically
run horizontally in a submarine mode. They also differ in their casting
methodology, as agarose sets thermally, while polyacrylamide forms in a
chemical polymerization reaction.
Agarose gels are made from the
natural polysaccharide polymers extracted from seaweed. Agarose gels
are easily cast and handled compared to other matrices because the gel
setting is a physical rather than chemical change. Samples are also
easily recovered. After the experiment is finished, the resulting gel can
be stored in a plastic bag in a refrigerator.
Applications

 Estimation of the size of DNA molecules following restriction enzyme

digestion, e.g. in restriction mapping of cloned DNA.
 Analysis of PCR products, e.g. in molecular genetic
diagnosis or genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern transfer, or of
RNA prior to Northern transfer.
Gel electrophoresis is used in forensics, molecular
biology, genetics, microbiology and biochemistry. The results can be
analyzed quantitatively by visualizing the gel with UV light and a gel
imaging device. The image is recorded with a computer-operated
camera, and the intensity of the band or spot of interest is measured and
compared against standard or markers loaded on the same gel. The
measurement and analysis are mostly done with specialized software.
Depending on the type of analysis being performed, other techniques
are often implemented in conjunction with the results of gel
electrophoresis, providing a wide range of field-specific applications.
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