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Transcript of advancement in DNA Technology
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INTRODUCTION
The beginning of the 21ist century has witnessed a revolution in our
knowledge of the DNA sequences of various organisms, the most notable example being the
sequencing of the human genome in 2003. Analysis of genomic DNA will enable us to learn a
great deal about evolution, the relationship between different organisms, the mechanisms by
which genes are controlled, susceptibility to disease, and the hidden languages within the DNA
sequence. The sequencing of an entire genome is not yet, however, a routine technique, and other
Methods of Genetic Analysisare used to quickly and effectively analyze DNA samples. These
methods, and technologies such as DNA fingerprinting, have also transformed forensic science.
The DNA typing methods shows a mark able difference from 1985 (since its discovery by DR
Sir Alec Jeffrey) to till date. The past 18 years have seen tremendous growth in the use of DNA
evidence in crime scene investigations and paternity testing. This rapid growth is always
accompanied with the revolutions in computer technology. Established methods of DNA
sequencing, genetic and forensic analysis all depend on the use of labelled oligonucleotideand
deoxy- or dideoxy-nucleoside triphosphates, and require a DNA polymerization step. This can be
the polymerase chain reaction (Genetic analysis inSTR analysis)1, a single nucleotide
extension (Mini-sequencing inSNP analysis)2, or a combination of polymerization and DNA
chain termination (Sanger sequencing)3.
.
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DNA TYPING CHRONOLOGY
1985 Sir Alec Jeffreys develops multilocus RFLP probes.
1986 DNA testing goes to public through celmark case of Colin pitch fork.
1988 FBI begins DNA casework with single-locus RFLP probes.
1989 DNA detection by gel silver-staining,slot blot, and reverse dot blots.
1990 Population statistics used with RFLP methods are questioned, PCR starts
With DQA-1.
1991 Fluorescent STR markers first described; Chelex extraction.
1992 FBI starts casework with PCR-DQA1, Capillary arrays first described.
1993 First STR kit available, sex-typing (amelogenin) developed.
First STR results with CE.
1995 ABI 310 Genetic Analyzer and Taq Gold DNA polymerase introduced.
National DNA Database developed by UK 18 loci.
1996 FBI starts mtDNA testing, first multiplex STR kits become available.
1997 13 core STR loci defined, Y-chromosome STRs described.
1998 FBI launches national Combined DNA.
2000 SNP hybridization chip developed, Multiplex STR kits are validated .
ABI 3700 96-capillary array used.
2001 Identifier STR kit released with5-dye chemistry ABI 3100 Genetic
Analyzer.
Year Forensic DNA science and application
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2002 FBI mtDNA population database released-STR 20plex published.
2003 Human genome project completed. ENCODE PROJECT starts.
2004 454 Gs-80 pyro sequencing used. Next GEN starts based on Sanger
Dideoxy chain termination
2005 solexia/illumina sequencing starts.
2006 ABISOLiD sequencing
2007 Roche 454 titanium /Illumina GAIIx used sequencing.
ENCODE completed
2009 Illumina GAIIx, SOLiD 3.0
2010 Illumina Hi-Seq2000
NEXT GEN
2013
454/SOLiD/SolexiaHUMAN GENOME
2005
Y-STRs
NEXT GEN
developed
2004
2000
CE is fairly routine
RFLP
FIRST STR
Historical Perspective on DNA Typing
developed
UK NationalDatabase launched
First commercialMultiplexes
2002
Identifiler 5-dye kit
And ABI 3100
PowerPlex
mtDNA1990
FSS
1992
19961994
1998
Capillary electrophoresis
of STRs first described
CODIS loci
PCR developed
(Dot blot)1985 Multiplex STRsDQA1 & PM
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THE BEGINNING OF THE REVOLUTION
DNA analysis constitutes the most significant aspect of biotechnology related to forensic
science. Since it was first introduced in the mid-1980s, DNA analysis (formerly called DNA
Fingerprinting, but now increasingly referred to as DNA Typing or DNA Profiling) has
revolutionized forensic science like no other technique has, especially in the area of
identification of individuals. The technique was first described in 1985 by Dr. Alec Jeffreys, a
geneticist in the University of Leicester5. He discovered that certain regions of human DNA
contained sequences that repeated over and over contiguously, and that the number of such
repeats differed from individual to individual. By developing a technique to examine the length
variation of these repeat sequences, Dr. Jeffreys devised the ability to fix the identity of
individuals with a high degree of certainty. The technique developed by him came to be called
restriction fragment length polymorphism (RFLP). This really triggered the era of forensic
biotechnology, which has since moved at an amazing pace, and continues to do so, impacting
virtually every area of forensic investigation of serious crimes such as homicide, rape, and
assault.
DNA level individual people are 99.9% identical
1 out of 1000 base pairs differs.
Some DNA differences lead to differences in appearance.
Some differences are found in non-coding DNA.
There are repeats of short nucleotide sequences which are known as VNTRs (Variable
Number Tandem Repeats).
There are dozens or hundreds of alleles in a given population each person having two alleles,
one on each chromosome.
Identification is made based on the VNTRs found in junk DNA.
DNA Information
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1. DNA is isolated from the tissue sample.
2. DNA is then cut (at the molecular level) using an enzyme. The restriction enzymes areable to break apart the DNA molecule in a specific place. There are over 100 different
types of restriction enzymes but some are used commonly.
EcoRI came from bacteria called E.coli; HIND III came from Hemolphilous influenza,strain d
3. These enzymes cut the DNA in areas which are the same in everyone (not the junk DNAwhich varies in different individuals)
4. HIND III recognizes the DNA sequences6
5AAGCTT33TTCGAA5
and it cuts between the two As leaving
5AGCTT
TTCGA 5
5. This sequence is found frequently in the human genome, therefore HIND III cuts the
DNA into many pieces
6. DNA is then separated by loading it into an apparatus which will allow electricity to runthrough a gel plate holding the DNA sample. Different sized pieces of the DNA willseparate and a banding pattern will form allowing identification of matching DNA to
occur.
7. Fragments are separated by size
Wells (Negative End) are filled with DNA
DNA has a negative charge
Small pieces of DNA move farther away from the wellas electricity is run through the
gel.
Note: Though RFLP was the initial method adopted for use, it is no longer the preferred approach
in majority of the forensic laboratories because, it requires good amount of non-degraded DNA (~
1.0 g) which is often very difficult to obtain from the crime scene. Moreover, it uses radiolabel
(Ethidium Bromide) which is hazardous and takes 1-2 weeks for the result to be processed7.
Techniques in RFLP
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The polymerase chain reaction (PCR) is a technique invented by kary mullis in 19808, used
widely in molecular biology, diagnostics, forensic science and molecular genetics, to amplify a
specific region (the amplicon) of and A sample. PCR can amplify a few molecules of a precious
DNA sample (e.g. at the scene of a crime) to produce large quantities of DNA, from 50 to over
25 000 base pairs in length. In PCR, two short oligonucleotide (PCR primers) are designed such
that each is complementary to the 3-end of one of the two target strands at the region to be
amplified: the two PCR primers define the amplicon. The region of the template bound by the
primers is amplified in a series of cycles.PCR requires a DNA polymerase enzyme. While all
organisms contain DNA polymerases, the polymerase that is used in PCR comes from the
thermophilic bacterium Thermus aquaticus. This Taq polymeraseis heat-resistant, meaning that
temperatures of up to 95 C can be used in PCR, conditions of low DNA duplex stability9.
In the first cycle, the double stranded target is separated into two single template strands by
heating to 95 C. It is then cooled to 55 C to allow the synthetic oligonucleotide primers to
anneal to the template strands with their 3' ends facing each other. The temperature is then
increased to 72 C, the optimum temperature for activity of the thermo stable Taq DNA
polymerase. The polymerase utilizes deoxyribonucleotide triphosphates (dNTPs) to extend the
primers along the length of the template producing two new double strands of DNA.
The second cycle of PCR is a repeat of the first cycle, and each newly synthesized single strand
also acts as a template for primer annealing and extension. The polymerase can only be extend
the DNA as far as the locus of the first primer, producing DNA duplexes of a specific length. In
all subsequent cycles amplification produces PCR products of a length specified by the loci of
the two primers, and these PCR products soon outnumber the original target molecules. In
theory, ncycles of PCR will produce 2nPCR products.
Polymerase chain reaction
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Real-time PCR is a variation on the PCR theme that combines normal PCR amplification of
DNA with simultaneous detection of the PCR product, usually in a single reaction tube. In PCR,
the amount of double-stranded DNA increases with each cycle. After multiple cycles of PCR,
there is a large increase in the amount of DNA. In real-time PCR (also called quantitative PCR,
or qPCR), an agent that binds to double-stranded DNA is added to the PCR reaction. As double-
stranded DNA is produced, the agent binds to the newly-synthesized DNA, and produces a signal
allowing the reaction to be monitored in real time. While the aim of PCR is the amplification of
DNA, the purpose of real-time PCR is the analysis of a DNA sample or reaction.
Fluorescent real-time PCR is a combination of PCR amplification and fluorescence detection. In
its simplest form, fluorescent real-time PCR involves the use of an organic dye that is fluorescent
only when bound to a DNA duplex. When such a dye is added at the beginning of a PCR
reaction an increase in fluorescence occurs as the number of DNA duplexes increases, and this is
indicative of successful PCR. SYBR Green is an example of a molecule that binds to double-
stranded DNA and becomes fluorescent on binding (the ds-DNA-dye complex is fluorescent).
SYBR GREEN
Note: TheSYBR-Green real-time PCR method has severe limitations as it is non-specific,
i.e. a positive result is obtained regardless of the nature of the PCR product. As PCR is
prone to arte facts such as primer-dimer formation, simple amplification using unselective
dyes is not always very informative, and probe-based methods provide more meaningful
results10
.
Real Time PCR
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When using PCR in human diagnostics it is important to be certain of the precise nature of the
product. Identification of a key sequence in the PCR product (the amplicon) can be achieved by
adding a fluorogenic DNA probe (a short synthetic oligonucleotide that is complementary to a
specific sequence in the PCR amplicon, and does not fluoresce unless it binds to the amplicon) to
the PCR reaction.When a DNA probe is used in real-time PCR, a positive signal is obtainedonly if the PCR amplicon contains the complementary sequence to the fluorogenic probe: the
fluorescent signal is sequence-specific. In general "fluorogenic" probes contain a fluorescentdyes and a fluorescence quencher. They are non-fluorescent in the absence of a target nucleic
acid because the quencher absorbs energy from the excited fluorophore, and this energy is
dissipated as heat or radiation at a higher wavelength.
Probe based Real Time PCR
Probe Based Real Time PCR
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The Taq Man assay is the most widely used real-time method for the analysis of PCR products,
and is used extensively in SNP analysis and mutation detection. A Taq Man probe consists of an
oligonucleotide labeled with a fluorophore at one end, e.g. 5-FAM (5-fluorescein), and a
fluorescent quencher at the other, e.g. 3-TAMRA. Excitation of fluoresce in at its absorptionwavelength of 495 nm would normally lead to fluorescence emission at 525 nm. However, this
falls within the broad absorption spectrum of the TAMRA dye which is in close proximity in the
Taq Man probe, so energy is absorbed by the TAMRA dye owing to fluorescence resonance
energy transfer (FRET) and fluorescence is observed at the emission wavelength of TAMRA
(585 nm) rather than at the emission frequency of FAM
11
.
The Taq Man assay
The Taq Man assay
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DNA fingerprinting depends on the analysis of short tandem repeats (STRs), short repeating
patterns of two or more nucleotides (e.g. (CA)nor (ACGT)n, where nis several hundred). For
example, in the sequence CGTCAGCACACACACACACACACACACACACATGGCGTG,
the dinucleotide CAis repeated 13 times (n= 13).
Tens of thousands of different short tandem repeats, or microsatelliteshave been identified in
the human genome. STRs are observed at the same positions on chromosomes (loci) in different
members of the population, but the number of repeats (n) varies between individuals. This
variation in number of repeats is an example of polymorphism.
STR analysis uses PCR to measure the number of repeats at specific loci. Primers bind to the
DNA at specific STR loci and, are extended by PCR. The length of the PCR product depends on
the number of repeats. If the PCR primers are labeled, the PCR products will be labeled,
allowing the products to be detected at the end of the reaction. For each STR locus, there will be
two PCR products (one for each of two alleles)12
.
The simultaneous analysis of multiple different STR loci enables a unique profile of an
individual to be built up. Several PCR reactions are carried out simultaneously in a single tube at
different STR loci, giving several products (two for each locus). The following components are
required.
A DNA sample, e.g. a single human hair from the scene of a crime, or buccal cells from a
mouth scrape of a suspect.
Two oligonucleotide PCR primers: one primer labeled at the 5-end with32
P, and one
unlabelled reverse primer
A thermo stable DNA polymerase
Four deoxynucleoside triphosphates: dATP, dGTP, dCTP, dTTP.
When the labeled PCR products are run on a polyacrylamide gel, they separate according to size.
The result is a "DNA ladder" that is characteristic of an individual.
Short Tandem Repeats
STR Analysis
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DNA Fingerprinting By STR
The use of multiple loci provides a very high degree of certainty that no two individuals in a
population will have the same profile (unless they are identical twins). Some current forensic
systems use 10 (e.g. United Kingdom) or 13 (e.g. United States) and others 16 STR loci . Kits
containing PCR primers for the standard STR loci are sold commercially.
13 CODIS Core STR LociWith Chromosomal Positions
13
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
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In a more modern variant of STR analysis, the PCR primers are labeled with fluorescent dyes.
Primers for different STR loci are labelled with different fluorescent dyes, adding a second
dimension to the assay .As it has so far been possible to develop only a limited number of
fluorescent dyes with well-resolved spectral characteristics, three different fluorescent dyes are
typically used.
Fluorescent STR Analysis
NOTE: Conventional STR multiplex analysis works best where there is at least 1ng of good
quality DNA present and fewer than 28 PCR cycles are required to generate sufficient material
for a full PCR profile. However, many forensic samples contain much lower levels of DNA than
this and/or the DNA is degraded and in these circumstances a different approach is required.
Low copy number STR analysis (LCN - STR) is employed where there is less than 100 pg
DNA and despite the miniscule amounts of DNA present a full STR profile can be generated.
Some workers consider that defining LCN - STRin terms of the amount of DNA present in the
sample is not appropriate and prefer to consider it to be an approach adopted for the analysis of
results that occur below the stochastic threshold (i.e. the point below which their interpretation of
peaks or bands would be considered unreliable) using normal techniques. Using LCN - STR, it is
possible to obtain a full profile from the DNA of a single cell. LCN - STRmay employ up to 60
PCR cycles and although it is extremely sensitive the results need to be interpreted with care,
especially where the DNA of two or more people is present14.15
.
Fluorescent STR Analysis
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Since most DNA applications in the early years had been developed for the specific detection of
human DNA,only a few VNTRs of invertebrate DNA were known. This limitation was
overcome by a new technique that could be used on virtually any organism: randomly amplified
polymorphic DNA (RAPD). In this method, non-specific primers are used that can amplify many
regions of a sample DNA at once. The resulting PCR products are separated by electrophoresis,
and a band or peak of a particular length can be considered a locus even though it is not
known what portion of the sample DNA it represents. RAPDs can allow up to 100 or more loci
in one PCR. Since the high number of amplified RAPD loci can render the sorting of informative
PCR polymorphisms from non-informative ones difficult or confusing, specialised
electrophoresis unit and software programme must be used. In the forensic area, RAPD has
special importance in the entomological investigation of decaying corpses16
.
RAPD Profile
Randomly Amplified Polymorphic DNA
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In comparison to nuclear DNA, mitochondrial DNA (mtDNA) has some significant advantages
in forensic investigations. Firstly, it is present in high copy number, and can provide better
results when nuclear DNA is scanty, e.g., analysis of hair shafts, teeth, skin, etc.Secondly,
mtDNA is transmitted exclusively maternally to the offspring without undergoing
recombination. This clonal inheritance is of great use in identity testing because it allows direct
comparison of DNA sequences of relatives with the same maternal lineage, without the
ambiguities caused by meiotic shuffling and the mixing of nuclear genes.
In fact, when the sample sequence is compared to that of a reference person, the possibility of a
maternal relationship can be assessed. One significant disadvantage of mtDNA has been that
compared to nuclear DNA, the genome organization is very compact and, therefore less
polymorphic: over 90.0%of the genome is coding, introns are lacking, intergenic sequences are
very small or absent, and repetitive classes of DNA are relatively uncommon. For forensic DNA
testing, the most extensively studied region of mtDNA has been the non-coding DNA replication
control region (D-loop), located between the genes for tRNA-Pro and tRNA-Phe, at positions
16,024 to 576. mtDNAhas been used with great success in the forensic analysis of bones and
historical or ancient remains. However, amplification of mtDNA D-loop fragments with a length
of 200 bp or more from ancient and even from fairly recent biological samples, can lead to
erroneous results. Use of short PCR fragments for the analysis of mtDNA from shed hair, in
Mitochondrial DNA Testing
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combination with competitive PCR assay to determine the state of degradation, should improve
the reliability of forensic mtDNA analysis considerably. Due to the erroneous database
collection, the validity of sequence analysis of the mtDNA-loop hypervariable regions for
anthropological information about the maternal lineage has been questioned in many cases. To
avoid this, recommendations and guidelines have been proposed for the validity of mtDNA
sequence analysis and their interpretation in the forensic context17
.
Since heteroplasmy(same individual harboring more than one mtDNA sequence) is a potential
drawback to forensic mtDNA analysis, newer methods have focused on overcoming this problem
by enhancing detection capability of this phenomenon, for e.g., denaturing gradient gel
electrophoresis (DGGE). Several other technologies are also now being applied to mtDNA
analysis to make it more popular among the forensic community, including Mass
spectrometry18
, Microchip instrumentation19
, and Molecular beacon analysis20
.
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There has been an increasing interest among forensic investigators, in Y-chromosome markers,
not only for gender determination, but also for identity fixation. Y-chromosome markers are
useful for discriminating male DNA from female DNA in forensic situations such as sexual
assault, when a vaginal swab is submitted for DNA analysis. However, the amplification of Y-
chromosomal STRs is also known to result in the formation of artefactual amplification products,
mainly due to insufficient PCR specificity. This is a major drawback of the method, as both the
sensitivity as well as the correct Y-STR interpretation are affected. The addition of a PCR
enhancer to the reaction master-mix is claimed by some investigators to result in significant
increase of specificity of Y-STR typing-STRs are also useful for tracing paternal lineages, just as
mtDNA is used to match maternal lineages.21.22
YChromosome STR
Y-Chromosome STR
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Single nucleotide polymorphisms (SNPs) represent the ultimate in the trend toward smaller DNA
fragments. Recent advances in SNP research have raised the possibility that these markers could
replace the forensically established STRs. SNPs are more numerous than other polymorphisms,
and occur in coding and non-coding regions throughout the genome. They are single base-pair
changes in the DNA sequence, which can be detected by sequencing, RFLP-PCR or single-
strand conformational polymorphism (SSCP) techniques. A set of SNPs decoding identification
of an individual demands only a short stretch of DNA (
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An alternativeto SNPsfor the identification of racial characteristics from DNA is the analysis
of mobile element insertion polymorphisms based on short interspersed elements (SINEs). The
commonest class of these are the so - called Alu elementsthat are about 300 nucleotides long25
.
Most Alu elements are fixed at a particular locus but a few subfamilies are polymorphic for
insertion presence/absence and can be used to determine genetic relationships between
populations .Alu family of short interspersed nuclear elements (SINEs) is distributed throughout
the primate lineage and is the predominant SINE within the human genome. The Alu family has
spread throughout the genome by an RNA-mediated transposit ion process known as retro
position and is present in the genome in extremely high copy number (in excess of 500,000
copies per haploid human genome). The majority of Alu family members are pseudo gene
products of a single master gene. Sequence divergence in the master gene and its progeny
occurs with time, resulting in subfamilies. Young Alu subfamilies are polymorphic and are
present or absent on given chromosomes. The first appearance of the Alu insertion represents the
beginning of the family tree, and can be used as a molecular clock to estimate the time that
family or subfamily arose. Thus, unlike other forensic DNA markers, the distribution of Alu
insertions, and possibly long interspersed nuclear elements (LINEs) and other SINEs loci, permit
tracing of population ancestral heritages. Information about the likely ethnicity of the sources of
the sample is one piece of information that investigators may use when pursuing leads based on
the genetic analysis of crime scene evidence.26
Alu Repeats
Mobile Element Insertion polymorphism
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DNA microarray technology (also known as DNA arrays, DNA chips or biochips) represents one
of the latest breakthroughs and indeed major achievements in experimental molecular biology.
This novel technology, which started to appear during the second half of the 1990s,
Oligonucleotide can be chemically attached to the surface of materials such as glass or silicon,
on which they form small "spots" of around 100 m (104
m) in diameter. Large numbers of
oligonucleotide can be laid down on a single slide to form a microarray, and single strands of
fluorescently-labelled DNA (labelled PCR products or cDNA) can be captured by hybridization.
(cDNA is single stranded DNA complementary to the RNA from which it is synthesized by
reverse transcription. It gives indirect information on the nature of the various RNA messages
expressed in a cell (expression analysis)). If such a microarray contains 1000 spots then in theory
it is possible to hybridize a unique complementary nucleic acid sequence to each spot. The
identity of the DNA sequence is deduced from the location of the spot to which it hybridizes
using a fluorescence scanner. The fluorescent label attached to the captured nucleic acid strand
can be added by a number of different methods. PCR products can be labelled at the 5 -end
simply by using a PCR primer containing a 5-fluorescent dye. PCR primers can be labelled with
multiple fluorophore, but these tend to quench each other and also inhibit the PCR reaction. A
better way to introduce multiple labels into the PCR product is to use fluorescently labelled
deoxynucleoside triphosphates in the PCR or reverse transcriptase reaction (e.g.fluorescein-
labelled dT). However, the efficiency of the PCR reaction may be compromised by the chemical
modification on the heterocyclic base, which can inhibit the Taq polymerase. A carefully
determined mixture of unlabelled and labelled deoxynucleoside triphosphates must therefore be
used, and it is rare to achieve labeling densities greater than one fluorophore per 30 nucleotides.
Microarray assays can also be carried out in the reverse format by attaching individual PCR
DNA Microarray
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products to the slide as discrete spots and probing with a pool of fluorescently labeled
oligonucleotide.27
DNA microarrays are useful in high-throughput mutation, SNP and gene expression analysis
because very large numbers of DNA strands can be attached to a single array. Microarrays are
amenable to automation by robotic systems, allowing very high throughput. However, they
present challenges owing to some undesirable chemical and biophysical properties of molecules
on surfaces. Firstly, it is difficult to create very dense arrays. A spot size of 100 m is
achievable, but smaller spots (e.g. 1 m) would allow far higher numbers of spots per array,
permitting the use of smaller volumes of solution-phase DNA and greater throughput. Secondly,
the hybridization of complementary DNA molecules on a surface is not nearly as efficient as
solution hybridization. To make the system workable, the properties of the surface and the nature
of the linker between the surface and the attached DNA must be carefully controlled.
DNA Microarrays
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In situ hybridization (ISH), which allows the identification and visualization of specific DNA
sequences on chromosomes using radioactive labels, are discussed in. The synthesis and
applications of chemically modified oligonucleotide. Fluorescence in situ hybridization (FISH),
which extends ISH by employing fluorescence-based detection and visualization by fluorescence
microscopy, is an important tool in genetic analysis. The principle of FISH lies in the annealing
of a labelled probe to its complementary strand within the chromosomes of fixed cells or tissues,
followed by detection of the fluorescent label. The probes (DNA or RNA) are usually prepared
by one of three polymerase enzyme-based methods (nick translation, random priming or PCR)
which allow the incorporation of fluorescently-labelled deoxynucleoside triphosphates. An
average incorporation level of one fluorescent label per 30 nucleotides is typical. The length of a
DNA probe can be between 100 bp and 1000 bp. Longer probes increase non-specific
background fluorescence but short probes can be difficult to detect owing to insufficient
hybridization and low levels of labeling. It is important that the target is accessible to the probe
and must be retained in situ, not degraded by nuclease enzymes. Visualization limits span from
an entire chromosome to a 40 kb chromosomal section.28
Radio Active Chips
Fluorescence In Situ Hybridization (FISH)
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All this has been possible because of methods developed by Fred Sanger in Cambridge over 30
years ago. Sanger developed a novel method of DNA sequencing (the dideoxy method) for
which he was awarded his second Nobel Prize, in 198029
.
Sanger's dideoxy method of DNA sequencing was the first method that was used routinely for
sequencing of DNA in the laboratory. The following components are required for Sanger
sequencing:
A DNA template to be sequenced
An oligonucleotide primer labelled at the 5-end with32
P
A DNA sequencing polymerase
Four deoxynucleoside triphosphates: dATP, dGTP, dCTP, dTTP
Four dideoxy nucleoside triphosphates (nucleoside triphosphates lacking both 2- and 3-
hydroxyl groups): ddATP, ddGTP, ddCTP, ddTTP .
Sanger sequencing is a modified form of DNA Raplication.The primer hybridizes to a specific
locus on the template and the polymerase binds and incorporates nucleotides to assemble a
reverse complimentary copy of the template.30
Dideoxy Sequencing
DNA Sequencing
Sanger Dideoxy DNA Sequencing
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In the automated high-throughput fluorescent version of Sanger sequencing, an unlabelled
oligonucleotide primer is used, along with a thermostable DNA polymerase, four normal
deoxynucleoside triphosphates, and four dideoxy nucleoside triphosphates with different
fluorescent labelson them.31
Now onlyonesequencing reaction is necessary because termination in ddA gives the DNA
fragment a particular fluorescent colour, ddG a different colour, ddC a third colour and ddT a
fourth colour. The nature of the fluorescent dyes depends upon the DNA sequence used, but the
basic requirement is four dyes with well-resolved fluorescence emission spectra. A common
system uses FAM, JOE, TAMRA and ROX as the four dyes.
Fluorescence Dideoxy Sequencing
Existing DNA sequencing methods (including next-generation sequencing) are not able to detect
modified bases. With the recent surge in interest in Epigenetic, the failure to distinguish between
cytosine and 5-methylcytosine (both of which form Watson-Crick base pairs with guanine) is a
serious drawback of current sequencing technologies.
Fluorescence Dideoxy DNA Sequencing
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Bisulfite (HSO3-) deaminates unmethylated cytosine to uracil, but does not react with methyl
cytosine (Figure 7). This provides a method for sequencing DNA containing 5-methylcytosine
bases. The DNA is sequenced before and after bisulfate treatment: any change from cytosine to
uracil is ascribed to unmethylated cytosine, while cytosine bases that remain after bisulfite
treatment are assumed to be methylated in the original sample. This provides a method for
sequencing DNA containing 5-methylcytosine bases. The DNA is sequenced before and after
bisulfite treatment: any change from cytosine to uracil is ascribed to unmethylated cytosine,
while cytosine bases that remain after bisulfite treatment are assumed to be methylated in the
original sample.32
Bisulfite Sequencing
Bisulfite Sequencing
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Three main companies occupy the bulk of the next generation sequencing market.
454/pyrosequencing (Roche).
SOLiD (Applied Biosystems).
Solexia Illumina.
However, increased bases sequenced at a reduced cost have always been desired. For this reason
new theories were developed in the late 90s. These have come to fruition in the last 5 years or so
with advances in chemical and physical technology. Thus, we have now entered the next
generation era of sequencing. The analysis techniques are always being improved with new
algorithms developed all the time. In addition, we are now seeing newer sequencing theories
being developed, so called, next-next generation sequencing.
National Human Genome Resource Institute
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Over the recent years, DNA profiling has become a cutting-edge crime investigating technique
and an invaluable instrument in search of justice. With its capability to implicate or eliminate,
DNA evidence may play a significant role at various points throughout the life of a criminal
case, from initiation of a case to post-conviction confirmation of the truth. There are few
techniques in the history of forensic science that have thoroughly scrutinized and validated than
forensic DNA typing. The introduction of this new technology therefore, would be considered a
milestone in criminal investigation and legal system of our country.
The main aims of the new technology can be summarized:
To enable faster processing.
To reduce costs.
To improve sensitivity
To produce portable instruments.
To de-skill and to automate the interpretation process.
To improve success rates.
To improve quality of the result and to standardize processes.
To develop the kits for individual identification.
The next few years will probably see a new revolution as this new technology comes of age with
advances in the next generation era of sequencing and becomes widely available.
Conclusion
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7/27/2019 advancement in DNA Technology
27/28
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7/27/2019 advancement in DNA Technology
28/28