This ppt is about Huma Genetics

1. University of The Gambia
Human Genetics Bio304
ASSIGNMENT: Techniques used in Forensic Science;
DNA Isolation, DNA Extraction PCR, Gel Electrophoresis
and Digestion and DNA Restriction.

2. Table of Content
Introduction to forensic science
Literature review
DNA extraction
DNA isolation
PCR( polymerase Chain Reaction)
Gel electrophoresis
DNA restriction and digestion

3. Introduction To Forensic
Science
What Is Forensic Science?
Forensic science is the application of sciences such as
physics, chemistry, biology, computer science and engineering
to matters of law. Forensic science can help investigators
understand how blood spatter patterns occur (physics), learn
the composition and source of evidence as drugs and trace
materials (chemistry) or determine the identity of an such
unknown suspect (biology).
Forensic science plays a vital role in the criminal justice
system by providing scientifically based information
through the analysis of physical evidence. During an
investigation, evidence is collected at a crime scene or
from a person, analyzed in a crime laboratory and then the
results presented in court. Each crime scene is unique,
and each case presents its own challenges.

4. Introduction to forensic
science
 Forensic science started about a century ago. But it was less
advance than present day forensic.
 The word forensic comes from the Latin term forensis,
meaning "of or before the forum". The history of the term
originates from Roman times, during which a criminal charge
meant presenting the case before a group of public
individuals in the forum. Both the person accused of the crime
and the accuser would give speeches based on their sides of
the story.
 This origin is the source of the two modern usages of the
word forensic – as a form of legal evidence and as a category
of public presentation. In modern use, the term forensics in
the place of forensic science can be considered correct, as
the term forensic is effectively a synonym for legal or related
to courts. However, the term is now so closely associated
with the scientific field that many dictionaries include the
meaning that equates the word forensics with forensic
science.

5. Introduction of forensic
science
The main purpose of forensic science is
to find, gather and analyze evidence that
can be use in court.
There are several techniques use in
gathering evidences in crime scenes.
Among the techniques used are the
collection of any source of material were
DNA can be extracted , isolated
quantified and arranged through different
mechanisms and procedures to compare
it with a suspect’s DNA.

6. What is DNA?
Deoxyribonucleic acid or DNA is a
molecule that contains the instructions
an organism needs to develop, live
and reproduce. These instructions are
found inside every cell, and are
passed down from parents to their
children.

7. DNA structure
DNA is made up of molecules called
nucleotides. Each nucleotide contains a
phosphate group, a sugar group and a
nitrogen base.
The four types of nitrogen bases are adenine
(A), thymine (T), guanine (G) and cytosine
(C). The order of these bases is what
determines DNA's instructions, or genetic
code.
 Human DNA has around 3 billion bases, and
more than 99 percent of those bases are the
same in all people, according to the U.S.
National Library of Medicine (NLM

8. Diagram of the structure of DNA

9. Diagram of the structure of DNA
with the bases.

10. Cont’…..introduction.
 In this research , we are tasked to look
into the techniques of forensic
science; DNA extraction, DNA
isolation, PCR, Gel Electrophoresis,
DNA digestion and Restriction.

11. Literature Review
 DNA is the heredity material found in
living organisms of all categories.
 This have enable living organisms to
make copies of themselves through cell
division.
 As a result, forensic scientist use this
amazing double helix structure DNA
(Watson and Crick) to find evidences to a
lot of criminal cases to be presented in
court.
 (Kelly m. Elkins ,forensic DNA
Biology,2013)

12. Literature Review
In order to study DNA, you first have to get it
out of the cell. In eukaryotic cells, such as
human and plant cells, DNA is organized as
chromosomes in an organelle called the
nucleus.
Bacterial cells have no nucleus. Their DNA is
organized in rings or circular plasmids, which
are in the cytoplasm.
The DNA extraction process frees DNA from
the cell and then separates it from cellular
fluid and proteins so you are left with pure
DNA. (Kelly m. Elkins ,forensic DNA
Biology,2013)

13. Cont’…….on literature review
Research has shown that DNA can be
obtained from different sources like solid
sources examples hair, bones papers
etc.
It can also be obtained from liquid like
blood, sliver etc.
DNA can as well be obtained through the
cells, tissues and organs of different
organisms ranging from humans,
bacteria, plants etc.
(Kelly m. Elkins ,forensic DNA
Biology,2013

14. Cont…. Literature review
The obtaining of DNA can be done
through processes like DNA extraction,
isolation, amplification and matching of
DNA fragments.
DNA extraction is simply how to obtain
DNA from its source.
To isolate DNA is to purify the DNA from
other materials.
To amplify DNA, is to quantify it to a
larger amount to be enough to run an
analysis on it.
(Kelly m. Elkins ,forensic DNA
Biology,2013)

15. Aims of this research
This Research aims at;
 looking at forensic science.
Techniques used in forensic science;
DNA Isolation, DNA Extraction, PCR
and Gel Electrophoresis.
Procedures used in the above
mentioned techniques.

16. What is DNA Extraction?
DNA extraction involves separating the
nucleic acids in a cell away from proteins
and other cellular materials.
Different methodologies widely used by
forensic DNA scientists include organic,
Chelex, or solid-phase extraction.
Post-extraction filtration is sometimes
used to concentrate low amounts of
recovered DNA sample. (John M. Butler,
in Advanced Topics in Forensic DNA
Typing: Methodology, 2012)

17. DNA extraction procedures
 The three basic steps of DNA extraction are 1) lysis, 2)
precipitation, and 3) purification.
 Step 1: Lysis
In this step, the cell and the nucleus are broken open to
release the DNA inside and there are two ways to do this.
First, mechanical disruption breaks open the cells.
 This can be done with a tissue homogenizer (like a small
blender), with a mortar and pestle, or by cutting the tissue into
small pieces.
 Mechanical disruption is particularly important when using
plant cells because they have a tough cell wall.
Second, lysis uses detergents and enzymes such as
Proteinase K to free the DNA and dissolve cellular proteins.
.

18. Cont’…..extraction
Step 2: Precipitation
When you complete the lysis step, the DNA
has been freed from the nucleus, but it is now
mixed with mashed up cell parts.
Precipitation separates DNA from this cellular
debris. First, Na+ ions (sodium) neutralize the
negative charges on the DNA molecules,
which makes them more stable and less
water soluble. Next, alcohol (such as ethanol
or isopropanol) is added and causes the DNA
to precipitate out of the aqueous solution
because it is not soluble in alcohol.

19. Cont…..extraction
Step 3: Purification
Now that DNA has been separated
from the aqueous phase, it can be
rinsed with alcohol to remove any
remaining unwanted material and
cellular debris.
 At this point the purified DNA is
usually re-dissolved in water for easy
handling and storage

20. Organic extraction
Organic extraction involves the addition
of and incubation in multiple different
chemical solutions; including a lysis step,
a phenol chloroform extraction,
an ethanol precipitation, and washing
steps.
Organic extraction is often used in
laboratories because it is cheap, and it
yields large quantities of pure DNA.
Though it is easy, there are many steps
involved, and it takes longer than other
methods.

21. Cont’……organic extraction.
It also involves the unfavorable use of
the toxic
chemicals phenol and chloroform, and
there is an increased risk of
contamination due to transferring the
DNA between multiple tubes.

22. Chelex extraction
Chelex extraction method involves adding the
Chelex resin to the sample, boiling the
solution, then vortexing and centrifuging it.
The cellular materials bind to the Chelex
beads, while the DNA is available in
the supernatant.
The Chelex method is much faster and
simpler than organic extraction, and it only
requires one tube, which decreases the risk
of DNA contamination.
Unfortunately, Chelex extraction does not
yield as much quantity and the DNA yielded
is single-stranded, which means it can only
be used for PCR-based analyses and not
for RFLP

23. Solid phase extraction
Solid phase extraction such as using
a spin-column based extraction method
takes advantage of the fact that DNA
binds to silica.
The sample containing DNA is added to
a column containing a silica gel or silica
beads.
The DNA binds to the silica, while the
rest of the solution is washed out.
The DNA can then be removed from the
silica using a buffer.

24. Cont’….solid phase extraction
This method is very easy and uses
only one tube; it also yields high
quantities of pure, high-quality DNA,
and the procedure can even be
automated.
Multiple solid phase extraction
commercial kits are manufactured and
marketed by different companies;
the only problem is that they are more
expensive than organic extraction or
Chelex extraction

25. Diagram of the different
extraction methods.

26. Importance of DNA extraction
DNA extraction is an important step in
diagnosis use.
It is also useful in the detection of
bacteria and viruses.
It also provide the chance to diagnose
diseases and other genetic disorders.

27. DNA Isolation
DNA isolation is a process of
purification of DNA from sample.
DNA isolation is part of the DNA
extraction processes
The first isolation of DNA was done in
1869 by Friedrich Miescher. Currently it
is a routine procedure in molecular
biology or forensic analyses.
Isolation basically have to do with
removing the impurities to make DNA
pure. This can be done through any of
the DNA extraction methods.

28. Cont’….DNA Isolation
Breaking the cell membranes open to expose
the DNA along with the cytoplasm within (cell
lysis).
◦ Lipids from the cell membrane and the nucleus
are broken down with
detergents and surfactants.
◦ Breaking proteins by adding a protease
(optional).
◦ Breaking RNA by adding an Rnase (optional).
The solution is treated with concentrated salt
solution (saline) to make debris such as
broken proteins, lipids and RNA to clump
together.
Centrifugation of the solution, which
separates the clumped cellular debris from
the DNA.

29. Which method of DNA isolation
to be used?
The isolation method of choice is
dependent upon
The source of the DNA: blood, tissue,
bacterial, virus etc.
The final application: PCR, restriction,
sequencing, fingerprinting,
library construction etc.
The type of DNA: genomic vs. plasmid;
To a lesser extent the number of samples to
be processed
robotics/automation

30. Polymerase Chain Reaction
(PCR)
 What is PCR (polymerase chain reaction)?
After DNA extraction, the most common analysis is the PCR
(Polymerase Chain Reaction).
 Polymerase chain reaction (PCR) is a laboratory
technique that is used for amplifying a particular
segment of DNA. It is a laboratory procedure that can
be used to make many copies of a particular section of
DNA.
 PCR is a common tool that is used in molecular biology,
medical and biological research labs. It can be used in
the early stages of processing DNA for sequencing, for
detecting the presence or absence of a gene to help
identify pathogens, and generating forensic DNA
profiles from tiny samples of DNA.
 PCR was first developed in 1983 by an American
biochemist Kary Mullis. He was awarded the Nobel
Prize in Chemistry in 1993 for his pioneering work.

31. Reagents Used in PCR
Basically, there are five reagents used in PCR:
DNA template to be copied
PCR primers – they are short stretches of
DNA that initiates the PCR reaction. They
are designed to attach (anneal) to DNA
templates.
DNA nucleotides base – needed to construct
the new strands of DNA.
Taq polymerase enzyme – adds in the new
DNA bases
PCR buffer (magnesium and potassium) - to
ensure the right conditions for the reaction.

32. Procedure/Steps in PCR
PCR involves a process of heating and cooling called
thermal cycling, which is carried out by a machine.
There are three main stages involved in PCR:
 Denaturation:
In this stage, the DNA template is heated to 94˚C for
15 to 30 seconds. This breaks the weak hydrogen
bonds that hold DNA strands together in a helix,
allowing the strands to separate creating sing
stranded DNA.
 Annealing:
During this stage, the reaction is cooled to 50-65˚C for
20-40 seconds. This enables the primers to attach to
a specific location on the single stranded DNA
templates.
 Extension/Elongation:
This stage occurs at 72˚C. In this stage, the

33. Cont…PCR
With one cycle, a single segment of
double-stranded DNA template is
amplified into two separate pieces of
double-stranded DNA. These two pieces
of double-stranded DNA are then
available for amplification in the next
cycle.
 As the cycles are repeated, more and
more copies are generated and the
number of copies of the template is
increase exponentially.

34. A Diagram Showing PCR Steps

35. Template Strand

36. Cont’……..PCR
These steps are repeated up to 40
times. The times and temperatures
depend on how long the primers and
target segment are. For example,
longer segments require higher
temperatures and longer times to
denature.

37. Applications (importance) of
PCR
PCR is used in forensic sciences like DNA finger
printing, paternity testing and criminal
identification. A small quantity of original DNA is
needed, for example, a droplet of blood or a
single hair is sufficient for enough DNA to be
extracted from.
 PCR is an important technique in gene cloning,
which allows generations of large amount of pure
DNA from tiny amount of template stranded and
further study of a particular gene.
 PCR is useful in clinics to analyze specimens for
the presence of infectious agents, such as HIV,
hepatitis, malaria, anthrax, etc.
It is used in vaccine production by recombinant
DNA technology.

38. Cont…
PCR is used in studying mutations that occur
in many genetic diseases, such as cystic
fibrosis, sickle cell anemia, muscular
dystrophy, hemoglobinopathies, etc.
PCR is also essential in the Human Genome
Project (HGP) for determining the sequence
of the 3 billion base pairs in the human
genome.
PCR is used in evolutionary biology to
identify and study the relationships among
species (phylogenetic analysis).
PCR is used by paleontologists to amplify
DNA from extinct species or fossils such as
hair, bones, mummified tissues, etc.

39. Gel Electrophoresis
Separation of nucleic acids
 DNA molecules of different sizes can be separated by
gel electrophoresis.
 Larger molecules migrate more slowly than smaller
ones through the matrix of the gel.
 Gel electrophoresis includes:
Agarose
Polyacrylamide
Pulse field
 Agarose is polysaccharide which is extracted from
seaweed. It is used to separate DNA fragments of 300-
10,000 bp at 0.5-2%;
 The agarose form a solid matrix which allows DNA to
migrate through an electric field based on size;

40. Gel Electrophoresis
 The purpose of gel electrophoresis or “running a gel” is
to visualize whether or not your DNA extraction and/or
subsequent PCR reaction actually worked.
 Although PCR is supposed to only amplify a single pure
product, the reality is that you end up with a mix of
primer-dimers (primers binding to each other instead of
the template strand) and incorrect fragments in addition
to your desired product.
 Gel electrophoresis is used to sort DNA fragments by
size (number of base pairs). By comparing PCR
products to a “ladder” or a set of known standard base
pair lengths, you can estimate the length of the
fragments from your PCR and look for one that matches
the size of the product you were trying to amplify.

41. Reagent used in Gel
Electrophoresis
To do a gel electrophoresis, you will need the
following items:
Gel rig: this is a specialized piece of
equipment for casting your gel and doing an
electrophoresis.
PCR product: this is the mixture of DNA
fragments you want to sort.
DNA ladder: a solution of DNA molecules of
varying length that is used as a size
reference.
Buffer: this is used to make up the gel,
maintains the pH, and contains the ions
necessary to carry an electric charge.

42. Cont….on Gel
Electrophoresis
Agarose gel: this is a type of gelatin from
seaweed that will separate the DNA
fragments.
Loading dye: this dye is added to the
DNA sample to make it easier to handle.
DNA stain: this dye binds to DNA so the
bands can be seen in the
transilluminator. Examples include
Ethidium Bromide.
Transilluminator: this machine produces
UV, blue, or white light which makes the
DNA stain (and the DNA) glow. Different
sources of light correspond to different
stains

43. Procedures of Gel
electrophoresis
 DNA molecules carry an overall negative charge.
Since opposites attract, DNA is attracted to
positive charges, in this case, the (+) electrode in
the gel rig. To get to the (+) electrode, DNA has
to travel through a sheet of agarose gel.
 Smaller pieces are able to travel through the gel
faster than long pieces. Fragments of the same
length travel at the same speed and form clear
bands in the gel.
 When the gel is finished running, it is soaked in a
DNA Stain, a chemical that does two things: 1)
bind to double-stranded DNA and 2) glow under
UV, blue, or white light.

44. Cont…..
 Visualization of DNA
 The DNA can be visualized by
staining with ethidim bromide
(EtBr).
 EtBr is an intercalating agent which
will insert itself within the bases of
the DNA and will exhibit
florescence under UV light.
 DNA will also bind EtBr
but to a lesser extent.

45. Electrode and gel well.

46. Full set of the gel apparatus

47. Images in Gel Electrophoresis

48. Polyacrylamide gels
Polyacrylamide gel:
Have smaller pores than agarose.
Can separate DNA fragments which
range in size from 10-500bp;
DNA fragments which differ in size by
one nucleotide can be separated from
each other.
Polyacrylamide gel electrophoresis is
also used to separate protein molecules.

49. DNA Restriction
restriction endonuclease, a protein
producedby bacteria that cleaves DNA at
specific sites along the molecule.
 In the bacterial cell, restriction
enzymes cleave foreign DNA, thus
eliminating infecting organisms.
 Restriction enzymes can be isolated
from bacterial cells and used in the
laboratory to manipulate fragments of
DNA, such as those that contain genes;
for this reason they are indispensible
tools of recombinant DNA
technology(genetic engineering).

50. Cont’……DNA Restriction
Restriction enzymes were discovered
and characterized in the late 1960s
and early 1970s by molecular
biologists Werner Arber, Hamilton O.
Smith, and Daniel Nathans.
The ability of the enzymes to cut DNA
at precise locations enabled
researchers to isolate gene-containing
fragments and recombine them with
other molecules of DNA—i.e to clone
genes

51. Diagram of how restriction
enzyme cuts nucleotide.

52. Procedure of the restriction
enzyme working.
Restriction Endonuclease scan the
length of the DNA , binds to the DNA
molecule.
 when it recognizes a specific
sequence and makes one cut in each
of the sugar phosphate backbones of
the double helix – by hydrolyzing the
phoshphodiester bond.
 Specifically ,the bond between the 3’
O atom and the P atom is broken.

53. Reference
 Aryal S. 2018. Polymerase Chain Reaction (PCR)-Principle,
procedure, Types, Applications and Animation. Microbiology
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(https://microbiologyinfo.com/polymerase-chain-reaction-pcr-
principle-procedure-types-applications-and-animation/)
 Yourgenome organization, 25 January 2016. Facts. What is PCR
(polymerase chain reaction)? [Accessed 13 March, 2019 at 12:20
pm]. Retrieved from (https://www.yourgenome.org/facts/what-is-pcr-
polymerase-chain-reaction)
 Karki G. 2017. Polymerase chain reaction (PCR): Principle,
procedure or steps, types and application. Online Biology notes.
[Accessed 13 March, 2019 at 12:48 pm]. Retrieved from
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 Editorial Team, 27 January 2019. Polymerase Chain Reaction
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from (https://laboratoryinfo.com/polymerase-chain-reaction-pcr/)

54. Reference
Geneticeducation.co.in>different_21octo
ber 2018.
Kelly m.Elkins,in forensic Dna Biology
2013
Dahm R(january 2006)discovering Dna :
human genetics
Advanced topics in forensic Dna by John
m. Butler, 2015,pages 159-182
Methods in cell biology,volume112,2012
page1-32 by leland j.Cseke, Joseph R.
Herdy
 U.S. National Library of Medicine (NLM

55. Reference
http://www.dnalc.org/ddnalc/resources
/electrophoresis.html
Dna restriction and electrophoresis
Diamantina institute-university of
…..https://www.di.uq.edu.au>sparq-
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 Biochemistry (2002), Freeman & Co.
Berg, J.M., Tymoczco, J.L., Stryer, L.

56. Group Members