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October 18, 2012 
VIRUS AND FORENSICS 
MANISH SURVE, PRADIP HIRAPURE 
Government Institute of Forensic Science, Mumbai 
What is a Virus? 
A virus is a small infectious agent that can replicate only inside the living cells of an organism. 
Viruses can infect all types of organisms, from animals and plants to bacteria and archaea. 
Viruses are constituted of two or three parts: the genetic material made from 
either DNA or RNA, long molecules that carry genetic information; a protein coat that protects 
these genes; and in some cases an envelope of lipids that surrounds the protein coat when they 
are outside a cell. Some viruses, like HIV, have in addition an external envelope derived 
from the plasma membrane of the host cell from which it came. 
Forensic virology is the study of viruses in a forensic context. 
Viruses can cause death, either as a natural process or as a criminal act (e.g. bioterrorism). These 
viruses can be natural or engineered. 
Forensic virology can be used to determine the type and strain of virus, and the source of an 
engineered virus. 
The phrase ‘trace-back investigation’ refers to the process of tracing a virus back to its source in 
order to find out where an infection has come from. 
Structure of Virus: 
The capsid and entire viral structure can be of four main types: 
1. Helical 
There is a capsomer coiled around a central axis to form a helical structure. This is a common 
structure seen in single stranded RNA viruses. Tobacco mosaic virus is a helical virus. 
2. Icosahedral 
These are near-spherical and this shape is adopted because the coat forms a closed shell. 
Rota virus has twelve capsomers and appear spherical. 
3. Envelope 
The virus is covered with a lipid membrane in a modified form of one of the cell membranes. 
The outer membrane is from the infected host cell and internal membranes from nuclear 
membrane or endoplasmic reticulum forming a lipid bilayer known as a viral envelope. This 
membrane is studded with proteins or receptors. 
4. Complex 
There is a capsid that is neither purely helical, nor purely icosahedral. There may be extra 
features like protein tails or a complex outer wall. Bacteriophages are examples of this type of 
viral structure. 
MANISH DEEPAK SURVE, MSc-I, 20 Page 1
October 18, 2012 
An array of viruses. (a) The helical virus of rabies. (b) The segmented helical virus of influenza. 
(c) A bacteriophage with an icosahedral 
herpes simplex virus. (e) The unenveloped polio virus. 
(f) The icosahedral human immunodeficiency virus with spikes on its envelope. 
How does virus function in a host cell? 
MANISH DEEPAK SURVE 
head and helical tail. (d) An enveloped icosahedral 
SURVE, MSc-I, 20 
Viruses lie around our environment all of 
the time just waiting for a host cell to come 
along. They can enter us through the nose, 
mouth or breaks in the skin 
find a host cell to infect. For example, cold 
and flu viruses will attack cells 
respiratory or digestive tracts. The human 
immunodeficiency virus (HIV), which 
causes AIDS, attacks the T 
immune system. 
Regardless of the type of host cell, all 
viruses follow the same basic steps in what 
is known as the lytic cycle (see figure): 
once inside, they 
that line the 
T-cells of the 
a. A virus particle attaches to a host 
cell. 
b. The particle releases its genetic 
instructions into the host cell. 
c. The injected genetic material recruits 
the host cell's enzymes. 
d. The enzymes make parts for more 
new virus particles. 
e. The new particles assemble the parts 
into new viruses. 
Page 2 
rticles
October 18, 2012 
f. The new particles break free from 
the host cell. 
Virus as Bioweapon: 
Biological agents are likely to be used by terrorists as weapons because: 
• They are capable of damaging populations, economies, and food supplies 
• They can be directed at a small group of people or an entire population 
• They can be used to attack people, economies and food supplies 
• They cause fear, panic and social disruption 
• Most of them are obtained from nature 
• Certain agents are inexpensive to make 
• They are easily made by relatively unsophisticated methods 
• They are invisible to the senses 
• Their effects may be delayed 
Various delivery methods are possible: 
· aerosol 
· envelope or package 
· food or water contamination 
Viruses can cause death or ill effects in the body, either as a natural process or as a criminal act 
(e.g. bioterrorism). These viruses can be natural or engineered. 
Viral agents were first used as weapons by British General Jeffrey Amherst in 1763 against the 
Native Americans during the French and Indian War. As a biological weapon, viral agents would 
most likely be distributed in aerosolized form. Unlike bacteria, antibiotic treatments are not 
effective therapies against viral infections. Inside a host, viruses may remain dormant for long 
periods before reviving to infect other hosts. 
Most common diseases caused by Virus used as bioweapon: 
1. Small pox: 
a. Infectious agent: Variola virus - Orthopox virus 
b. Declared eradicated in 1980, but stockpiles may exist 
c. Not naturally acquired. It can be disseminated as and inhaled as an aerosol 
d. Symptoms: Fever, muscular rigidity, headaches, and vomiting. Severe cases 
experience prostration and hemorrhage into skin and mucous membranes 
e. Rash appears after about three weeks; progresses from macules (initial skin lesions) to 
papules to pustular vesicles, to scabs 
f. Transmission: may occur person to person by respiratory droplets or skin inoculation. 
Highly contagious when rash appears 
g. Incubation period: 10 to 12 days 
h. Mortality: less than 1% in the minor form and 20 to50 % in the major form 
i. Treatment: supportive 
j. Prevention: vaccine 
2. VIRAL HEMORRHAGIC FEVERS: 
a. These are highly infectious viral illnesses caused by the Filoviruses (Ebola and 
Marburg), Arenaviruses (Lassa fever), Bunyaviruses (Congo hemorragic fever and 
Hantaviral disease), and Flaviviruses 
MANISH DEEPAK SURVE, MSc-I, 20 Page 3
October 18, 2012 
b. Symptoms: vary from one type to the next. They include: sudden onset of fever, muscle 
aches, headache, followed by vomiting, diarrhea, and rash and internal bleeding 
c. Complications: In severe forms, multiorgan failure occurs, primarily due to hemorrhagic 
and pulmonary complications 
d. Mode of transmission: handling infected wild animals, but may be used as an aerosol 
bioterrorist weapon 
e. Incubation period: 2 to 21 days 
f. Reservoir: gorillas and chimpanzees 
g. Transmission: some may be spread person to person by contact with body secretions 
h. Mortality: Ebola rates have reached 90% but vary 
i. Treatment: supportive 
j. Prevention: Avoid contact with infected monkeys or other animal hosts. 
List of some viruses can be used as bio weapons: 
• EBOLA 
• LASSA FEVER 
• INFLUENZA 
• VIRAL HEPATITIS 
• VIRAL HEMORRHAGIC FEVERS 
• HIV etc 
Techniques used for Detection and Investigation: 
A. PCR 
What is Polymerase Chain Reaction? 
It is a fast and inexpensive technique used to amplify small and targeted segments of DNA to 
produce million of copies, sometimes called "molecular photocopying" of a specific gene 
fragment. 
PCR Steps: 
PCR is a three-step process which is repeated in several cycles. The three steps are: 
1. Denaturation step: This step consists of heating the reaction to 90–95 °C. for 1 min. It causes 
DNA separation by disrupting the hydrogen bonds between complementary bases, yielding 
single strands of DNA. 
2. Annealing step: The reaction temperature is lowered to 50–65 °C allowing hybridization of the 
primers to the single-stranded DNA template. It require 1-2 min 
3. Extension/Elongation step: At this step, the Taq polymerase synthesizes a new DNA strand 
complementary to the DNA template strand by adding dNTPs that are complementary to the 
template in 5' to 3' direction. 
This process is repeated as many as 30 or 40 times, leading to more than one billion exact copies 
of the original DNA segment. The entire cycling process of PCR is automated and can be 
completed in just a few hours using a machine called a thermal cycler. 
MANISH DEEPAK SURVE, MSc-I, 20 Page 4
October 18, 2012 
B. Random Amplified Polymorphic DNA (RAPD): 
RAPD is based on the amplification of genomic DNA with single primers of arbitrary nucleotide 
Sequence. These primers detect polymorphisms in the absence of specific nucleotide sequence 
Information and the polymorphisms function as genetic markers and can be used to construct 
genetic maps. Since most of the RAPD markers are dominant, it is not possible to distinguish 
whether the amplified DNA segment is heterozygous (two different copies) or homozygous (two 
identical copies) at a particular locus. In rare cases, co-dominant RAPD markers, observed as 
different-sized DNA segments amplified from the same locus, may be detected. 
The basic technique of RAPD involves (i) extraction of highly pure DNA, (ii) addition of single 
random (arbitrary) primer, (iii) polymerase chain reaction (PCR), (iv) separation of fragments by 
gel electrophoresis, (v) visualization of RAPD-PCR fragments after ethidium bromide staining 
under UV light and (vi) determination of fragment size comparing with known molecular marker 
with the help of gel analysis software. A diagrammatic presentation of these steps is given in 
above Figure. It is important to note that RAPD technique requires maintaining strictly 
consistent reaction conditions in order to achieve reproducible profiles. In practice, band profiles 
can be difficult to reproduce between (and even within) laboratories, if personnel, equipment or 
conditions are changed. Despite these limitations, the enormous attraction of this technique is 
that there is no requirement for DNA probes or sequence information for primer design. The 
procedure involves no blotting or hybridizing steps. Another advantage is the requirement for 
only small amounts of DNA (10-100 ng per reaction).The RAPD markers have been used for 
detecting genomic variations within and between individuals species. Genetic diversity was 
evaluated by RAPD markers and RAPD has been used for estimation of microbial genetic 
diversity. 
C. T-RFLP (terminal restriction fragments length polymorphism): 
Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis is one fingerprinting 
methodology that can incorporate automated genotyping systems for laser detection 
offluorescently-labelled DNA fragments. The T-RFLP analysis technique involves the 
amplification of a gene of interest using fluorescent primers, followed by restriction 
endonuclease digestion, and automated analysis of the end or terminal restriction fragments. 
Because the 16S rRNA genes from all of the bacteria in the soil community are amplified, the 
resulting PCR products are gene copies of a similar length but with different internal sequences. 
The PCR products are cleaved with a restriction enzyme that recognizes and cleaves DNA at 
particular sequences. In the variable regions of the 16S gene, restriction sites occur in different 
places resulting in different length fragments. The more diverse the bacterial community in a 
sample, the greater the range of resulting fragments. The end, orterminal fragments are analyzed 
and the size and frequency of each fragment assessed to produce a profile or ‘fingerprint’ of the 
whole bacterial community in a soil sample. The T-RFLP method was originally developed by 
Avaniss-Aghajani et al. (1994) to identify Mycobacteria, but its potential to analyze variation 
between genes from a mixture of bacteria was first shown by Liu et al. Since then, T-RFLP 
analysis has been used in numerous studies to look at bacterial, archaeal and eukaryotic 
populations in many different substrates, and has been identified as a reproducible and accurate 
MANISH DEEPAK SURVE, MSc-I, 20 Page 5
October 18, 2012 
tool for community fingerprinting play a importance role in forensic investigation as a microbial 
forensic tool. 
SSCP (single stranded conformational polymorphism) 
Increasingly in the genomics field, researchers are requiring screening and sequence variation 
detection tools for large numbers of samples. SSCP analysis detects sequence variations (single-point 
mutations and other small-scale changes) through electrophoretic mobility differences. 
These variations can potentially cause conformational changes in the DNA molecules. Under 
non denaturing conditions and often reduced temperature, single-stranded DNA molecules can 
assume unique conformations that vary depending on their nucleotide sequences so the 
conformation (structural) vary from species to species because the genetic sequences vary from 
species to species and individual to individual. These conformational changes can result in 
detectable differences in mobility as illustrated in Figure below. In this application note we will 
review SSCP analysis, a technique that is widely used for sequence variation detection because 
of its simplicity and ease of use in forensic investigation to develop the microbial community 
profiling or to study the microbial profiling of crime related suspect persons and try to find out 
the location of crime. 
D. Amplified ribosomal DNA restriction analysis (ARDRA): 
Amplified ribosomal DNA restriction analysis (ARDRA) is a simple procedure in which a 
standard restriction digestion analysis is performed on PCR-amplified rDNA. This method is 
also known as RFLP (restriction fragment length polymorphism). In this method, PCR 
amplification of rDNA genes (16S, 23S, etc.) is first performed on a community sample. 
Following this, various restriction enzymes or combinations thereof are used to digest the 
amplified community DNA.The operating principle of this method is that divergences in the 
rDNA gene sequences of different species will create differences in restriction sites for various 
enzymes. If the correct restriction enzymes are used, what should emerge is a unique fingerprint 
for each species or strain .This digested DNA is run on a gel, producing a pattern of fragment 
sizes that is characteristic of the community. For single isolates or clones, the digests can be run 
on regular agarose. However in studies of complex communities, the large number of DNA 
fragments produced by this method can only be resolved using polyacrylamide gels. 
E. Amplified fragment length polymorphism (AFLP): 
AFLP analysis is a genetic mapping technique that uses selective amplification of a subset of 
restriction enzyme-digested DNA fragments to generate a unique fingerprint for a particular 
genome. First developed for plant studies, AFLP analysis is used for a variety of applications, 
such as: 
• Creation of genetic maps for new species 
• Determination of relatedness among species 
• Establishment of linkage groups in parentage disputed cases 
• Genetic diversity and molecular phylogeny studies in microbial forensic. 
The power of AFLP analysis derives from its ability to quickly generate large numbers of marker 
fragments for any organism, without prior knowledge of the genomic sequence. In addition, 
AFLP analysis requires only small amounts of starting template and can be used for a variety of 
genomic DNA samples. The AFLP procedure consists of two amplification steps: a low-level or 
MANISH DEEPAK SURVE, MSc-I, 20 Page 6
October 18, 2012 
preselective amplification, followed by a more selective amplification, which generates a set of 
fragments that can be used as the discriminatory marker set for a particular sample. 
F. Denaturing gradient gel electrophoresis: 
Denaturing gradient gel electrophoresis (DGGE) is a method that separates PCR-amplified 
rDNA according to differences in sequence G-C content, based on differential mobility through a 
DNA-denaturing gel. In this method, PCR-amplified DNA from taxonomically differentiated 
genes is run on a special polyacrylamide gel, which has embedded a gradient of DNA-denaturing 
compounds, usually urea and form amide. As DNA passes through a concentration gradient (i.e., 
from low to high) of denaturant, it comes under increasing pressure to separate into single 
strands. The DNA is unable to denature completely because of the presence of a GC clamp, 
which is included in one of the primers for the PCR reaction. It does, however, become 
increasingly denatured as it passes through the gel, which decreases its mobility. The DNA 
comes to rest when it is almost fully denatured. The position along the gradient at which this 
occurs is determined primarily by the relative proportions of G+C and A+T in a given amplicon, 
since G-C bonds are more difficult to denature than A–T bonds. Thus, differences in sequence 
between amplicons that result in differences in G-C content will cause DNA to migrate to 
different positions in the gel. Properly calibrated, DGGE is sensitive enough to detect even single 
base-pair differences between amplicons. DGGE is perhaps the most commonly used method of 
community characterization, in a manner similar to the other PCR-based genetic fingerprinting 
techniques. 
G. TGGE (Temperature gradient gel electrophoresis): 
There exists a variant of DGGE called temperature gradient gel electrophoresis (TGGE). TGGE 
operates on the same principles as DGGE, provides approximately the same degree of 
specificity, and possesses the same advantages and limitations. The only difference is that TGGE 
employs a gradient based on temperature (which also denatures DNA differentially depending on 
G-C content), rather than a gradient 
based on chemical denaturants. 
REFERENCE: 
http://www.biology-questions-and-answers.com/viral-infection.html 
http://www.biology-questions-and-answers.com/viruses.html 
http://www.intechopen.com/books/forensic-medicine-from-old-problems-to-new-challenges/ 
forensic-microbiology 
http://www.globalsecurity.org/wmd/intro/bio_viral.htm 
MANISH DEEPAK SURVE, MSc-I, 20 Page 7

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Viral forensics

  • 1. October 18, 2012 VIRUS AND FORENSICS MANISH SURVE, PRADIP HIRAPURE Government Institute of Forensic Science, Mumbai What is a Virus? A virus is a small infectious agent that can replicate only inside the living cells of an organism. Viruses can infect all types of organisms, from animals and plants to bacteria and archaea. Viruses are constituted of two or three parts: the genetic material made from either DNA or RNA, long molecules that carry genetic information; a protein coat that protects these genes; and in some cases an envelope of lipids that surrounds the protein coat when they are outside a cell. Some viruses, like HIV, have in addition an external envelope derived from the plasma membrane of the host cell from which it came. Forensic virology is the study of viruses in a forensic context. Viruses can cause death, either as a natural process or as a criminal act (e.g. bioterrorism). These viruses can be natural or engineered. Forensic virology can be used to determine the type and strain of virus, and the source of an engineered virus. The phrase ‘trace-back investigation’ refers to the process of tracing a virus back to its source in order to find out where an infection has come from. Structure of Virus: The capsid and entire viral structure can be of four main types: 1. Helical There is a capsomer coiled around a central axis to form a helical structure. This is a common structure seen in single stranded RNA viruses. Tobacco mosaic virus is a helical virus. 2. Icosahedral These are near-spherical and this shape is adopted because the coat forms a closed shell. Rota virus has twelve capsomers and appear spherical. 3. Envelope The virus is covered with a lipid membrane in a modified form of one of the cell membranes. The outer membrane is from the infected host cell and internal membranes from nuclear membrane or endoplasmic reticulum forming a lipid bilayer known as a viral envelope. This membrane is studded with proteins or receptors. 4. Complex There is a capsid that is neither purely helical, nor purely icosahedral. There may be extra features like protein tails or a complex outer wall. Bacteriophages are examples of this type of viral structure. MANISH DEEPAK SURVE, MSc-I, 20 Page 1
  • 2. October 18, 2012 An array of viruses. (a) The helical virus of rabies. (b) The segmented helical virus of influenza. (c) A bacteriophage with an icosahedral herpes simplex virus. (e) The unenveloped polio virus. (f) The icosahedral human immunodeficiency virus with spikes on its envelope. How does virus function in a host cell? MANISH DEEPAK SURVE head and helical tail. (d) An enveloped icosahedral SURVE, MSc-I, 20 Viruses lie around our environment all of the time just waiting for a host cell to come along. They can enter us through the nose, mouth or breaks in the skin find a host cell to infect. For example, cold and flu viruses will attack cells respiratory or digestive tracts. The human immunodeficiency virus (HIV), which causes AIDS, attacks the T immune system. Regardless of the type of host cell, all viruses follow the same basic steps in what is known as the lytic cycle (see figure): once inside, they that line the T-cells of the a. A virus particle attaches to a host cell. b. The particle releases its genetic instructions into the host cell. c. The injected genetic material recruits the host cell's enzymes. d. The enzymes make parts for more new virus particles. e. The new particles assemble the parts into new viruses. Page 2 rticles
  • 3. October 18, 2012 f. The new particles break free from the host cell. Virus as Bioweapon: Biological agents are likely to be used by terrorists as weapons because: • They are capable of damaging populations, economies, and food supplies • They can be directed at a small group of people or an entire population • They can be used to attack people, economies and food supplies • They cause fear, panic and social disruption • Most of them are obtained from nature • Certain agents are inexpensive to make • They are easily made by relatively unsophisticated methods • They are invisible to the senses • Their effects may be delayed Various delivery methods are possible: · aerosol · envelope or package · food or water contamination Viruses can cause death or ill effects in the body, either as a natural process or as a criminal act (e.g. bioterrorism). These viruses can be natural or engineered. Viral agents were first used as weapons by British General Jeffrey Amherst in 1763 against the Native Americans during the French and Indian War. As a biological weapon, viral agents would most likely be distributed in aerosolized form. Unlike bacteria, antibiotic treatments are not effective therapies against viral infections. Inside a host, viruses may remain dormant for long periods before reviving to infect other hosts. Most common diseases caused by Virus used as bioweapon: 1. Small pox: a. Infectious agent: Variola virus - Orthopox virus b. Declared eradicated in 1980, but stockpiles may exist c. Not naturally acquired. It can be disseminated as and inhaled as an aerosol d. Symptoms: Fever, muscular rigidity, headaches, and vomiting. Severe cases experience prostration and hemorrhage into skin and mucous membranes e. Rash appears after about three weeks; progresses from macules (initial skin lesions) to papules to pustular vesicles, to scabs f. Transmission: may occur person to person by respiratory droplets or skin inoculation. Highly contagious when rash appears g. Incubation period: 10 to 12 days h. Mortality: less than 1% in the minor form and 20 to50 % in the major form i. Treatment: supportive j. Prevention: vaccine 2. VIRAL HEMORRHAGIC FEVERS: a. These are highly infectious viral illnesses caused by the Filoviruses (Ebola and Marburg), Arenaviruses (Lassa fever), Bunyaviruses (Congo hemorragic fever and Hantaviral disease), and Flaviviruses MANISH DEEPAK SURVE, MSc-I, 20 Page 3
  • 4. October 18, 2012 b. Symptoms: vary from one type to the next. They include: sudden onset of fever, muscle aches, headache, followed by vomiting, diarrhea, and rash and internal bleeding c. Complications: In severe forms, multiorgan failure occurs, primarily due to hemorrhagic and pulmonary complications d. Mode of transmission: handling infected wild animals, but may be used as an aerosol bioterrorist weapon e. Incubation period: 2 to 21 days f. Reservoir: gorillas and chimpanzees g. Transmission: some may be spread person to person by contact with body secretions h. Mortality: Ebola rates have reached 90% but vary i. Treatment: supportive j. Prevention: Avoid contact with infected monkeys or other animal hosts. List of some viruses can be used as bio weapons: • EBOLA • LASSA FEVER • INFLUENZA • VIRAL HEPATITIS • VIRAL HEMORRHAGIC FEVERS • HIV etc Techniques used for Detection and Investigation: A. PCR What is Polymerase Chain Reaction? It is a fast and inexpensive technique used to amplify small and targeted segments of DNA to produce million of copies, sometimes called "molecular photocopying" of a specific gene fragment. PCR Steps: PCR is a three-step process which is repeated in several cycles. The three steps are: 1. Denaturation step: This step consists of heating the reaction to 90–95 °C. for 1 min. It causes DNA separation by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA. 2. Annealing step: The reaction temperature is lowered to 50–65 °C allowing hybridization of the primers to the single-stranded DNA template. It require 1-2 min 3. Extension/Elongation step: At this step, the Taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction. This process is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment. The entire cycling process of PCR is automated and can be completed in just a few hours using a machine called a thermal cycler. MANISH DEEPAK SURVE, MSc-I, 20 Page 4
  • 5. October 18, 2012 B. Random Amplified Polymorphic DNA (RAPD): RAPD is based on the amplification of genomic DNA with single primers of arbitrary nucleotide Sequence. These primers detect polymorphisms in the absence of specific nucleotide sequence Information and the polymorphisms function as genetic markers and can be used to construct genetic maps. Since most of the RAPD markers are dominant, it is not possible to distinguish whether the amplified DNA segment is heterozygous (two different copies) or homozygous (two identical copies) at a particular locus. In rare cases, co-dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, may be detected. The basic technique of RAPD involves (i) extraction of highly pure DNA, (ii) addition of single random (arbitrary) primer, (iii) polymerase chain reaction (PCR), (iv) separation of fragments by gel electrophoresis, (v) visualization of RAPD-PCR fragments after ethidium bromide staining under UV light and (vi) determination of fragment size comparing with known molecular marker with the help of gel analysis software. A diagrammatic presentation of these steps is given in above Figure. It is important to note that RAPD technique requires maintaining strictly consistent reaction conditions in order to achieve reproducible profiles. In practice, band profiles can be difficult to reproduce between (and even within) laboratories, if personnel, equipment or conditions are changed. Despite these limitations, the enormous attraction of this technique is that there is no requirement for DNA probes or sequence information for primer design. The procedure involves no blotting or hybridizing steps. Another advantage is the requirement for only small amounts of DNA (10-100 ng per reaction).The RAPD markers have been used for detecting genomic variations within and between individuals species. Genetic diversity was evaluated by RAPD markers and RAPD has been used for estimation of microbial genetic diversity. C. T-RFLP (terminal restriction fragments length polymorphism): Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis is one fingerprinting methodology that can incorporate automated genotyping systems for laser detection offluorescently-labelled DNA fragments. The T-RFLP analysis technique involves the amplification of a gene of interest using fluorescent primers, followed by restriction endonuclease digestion, and automated analysis of the end or terminal restriction fragments. Because the 16S rRNA genes from all of the bacteria in the soil community are amplified, the resulting PCR products are gene copies of a similar length but with different internal sequences. The PCR products are cleaved with a restriction enzyme that recognizes and cleaves DNA at particular sequences. In the variable regions of the 16S gene, restriction sites occur in different places resulting in different length fragments. The more diverse the bacterial community in a sample, the greater the range of resulting fragments. The end, orterminal fragments are analyzed and the size and frequency of each fragment assessed to produce a profile or ‘fingerprint’ of the whole bacterial community in a soil sample. The T-RFLP method was originally developed by Avaniss-Aghajani et al. (1994) to identify Mycobacteria, but its potential to analyze variation between genes from a mixture of bacteria was first shown by Liu et al. Since then, T-RFLP analysis has been used in numerous studies to look at bacterial, archaeal and eukaryotic populations in many different substrates, and has been identified as a reproducible and accurate MANISH DEEPAK SURVE, MSc-I, 20 Page 5
  • 6. October 18, 2012 tool for community fingerprinting play a importance role in forensic investigation as a microbial forensic tool. SSCP (single stranded conformational polymorphism) Increasingly in the genomics field, researchers are requiring screening and sequence variation detection tools for large numbers of samples. SSCP analysis detects sequence variations (single-point mutations and other small-scale changes) through electrophoretic mobility differences. These variations can potentially cause conformational changes in the DNA molecules. Under non denaturing conditions and often reduced temperature, single-stranded DNA molecules can assume unique conformations that vary depending on their nucleotide sequences so the conformation (structural) vary from species to species because the genetic sequences vary from species to species and individual to individual. These conformational changes can result in detectable differences in mobility as illustrated in Figure below. In this application note we will review SSCP analysis, a technique that is widely used for sequence variation detection because of its simplicity and ease of use in forensic investigation to develop the microbial community profiling or to study the microbial profiling of crime related suspect persons and try to find out the location of crime. D. Amplified ribosomal DNA restriction analysis (ARDRA): Amplified ribosomal DNA restriction analysis (ARDRA) is a simple procedure in which a standard restriction digestion analysis is performed on PCR-amplified rDNA. This method is also known as RFLP (restriction fragment length polymorphism). In this method, PCR amplification of rDNA genes (16S, 23S, etc.) is first performed on a community sample. Following this, various restriction enzymes or combinations thereof are used to digest the amplified community DNA.The operating principle of this method is that divergences in the rDNA gene sequences of different species will create differences in restriction sites for various enzymes. If the correct restriction enzymes are used, what should emerge is a unique fingerprint for each species or strain .This digested DNA is run on a gel, producing a pattern of fragment sizes that is characteristic of the community. For single isolates or clones, the digests can be run on regular agarose. However in studies of complex communities, the large number of DNA fragments produced by this method can only be resolved using polyacrylamide gels. E. Amplified fragment length polymorphism (AFLP): AFLP analysis is a genetic mapping technique that uses selective amplification of a subset of restriction enzyme-digested DNA fragments to generate a unique fingerprint for a particular genome. First developed for plant studies, AFLP analysis is used for a variety of applications, such as: • Creation of genetic maps for new species • Determination of relatedness among species • Establishment of linkage groups in parentage disputed cases • Genetic diversity and molecular phylogeny studies in microbial forensic. The power of AFLP analysis derives from its ability to quickly generate large numbers of marker fragments for any organism, without prior knowledge of the genomic sequence. In addition, AFLP analysis requires only small amounts of starting template and can be used for a variety of genomic DNA samples. The AFLP procedure consists of two amplification steps: a low-level or MANISH DEEPAK SURVE, MSc-I, 20 Page 6
  • 7. October 18, 2012 preselective amplification, followed by a more selective amplification, which generates a set of fragments that can be used as the discriminatory marker set for a particular sample. F. Denaturing gradient gel electrophoresis: Denaturing gradient gel electrophoresis (DGGE) is a method that separates PCR-amplified rDNA according to differences in sequence G-C content, based on differential mobility through a DNA-denaturing gel. In this method, PCR-amplified DNA from taxonomically differentiated genes is run on a special polyacrylamide gel, which has embedded a gradient of DNA-denaturing compounds, usually urea and form amide. As DNA passes through a concentration gradient (i.e., from low to high) of denaturant, it comes under increasing pressure to separate into single strands. The DNA is unable to denature completely because of the presence of a GC clamp, which is included in one of the primers for the PCR reaction. It does, however, become increasingly denatured as it passes through the gel, which decreases its mobility. The DNA comes to rest when it is almost fully denatured. The position along the gradient at which this occurs is determined primarily by the relative proportions of G+C and A+T in a given amplicon, since G-C bonds are more difficult to denature than A–T bonds. Thus, differences in sequence between amplicons that result in differences in G-C content will cause DNA to migrate to different positions in the gel. Properly calibrated, DGGE is sensitive enough to detect even single base-pair differences between amplicons. DGGE is perhaps the most commonly used method of community characterization, in a manner similar to the other PCR-based genetic fingerprinting techniques. G. TGGE (Temperature gradient gel electrophoresis): There exists a variant of DGGE called temperature gradient gel electrophoresis (TGGE). TGGE operates on the same principles as DGGE, provides approximately the same degree of specificity, and possesses the same advantages and limitations. The only difference is that TGGE employs a gradient based on temperature (which also denatures DNA differentially depending on G-C content), rather than a gradient based on chemical denaturants. REFERENCE: http://www.biology-questions-and-answers.com/viral-infection.html http://www.biology-questions-and-answers.com/viruses.html http://www.intechopen.com/books/forensic-medicine-from-old-problems-to-new-challenges/ forensic-microbiology http://www.globalsecurity.org/wmd/intro/bio_viral.htm MANISH DEEPAK SURVE, MSc-I, 20 Page 7