GENOMIC TYPING

Genetic Fingerprinting (also called DNA testing, DNA typing, or DNA profiling) is a technique
used to distinguish between individuals of the same species using only samples of their DNA.
Although two individuals /micro-organisms will have the vast majority of their DNA sequence
in common, DNA profiling exploits differences between individuals. Although the structure
of DNA is the same throughout all species of plants, animals and microorganisms, each
individual organism looks different. This is due to the order in which DNA base pairs are
sequenced.

Why is fingerprinting/typing important?

Ever since Koch discovered how to grow bacteria in pure culture, the laboratory has been an
integral component of epidemiologic studies of bacterial diseases. Over time, our ability to
discriminate among bacterial strains from the same species has increased, enhancing
outbreak investigations and surveillance, studies of the natural history of infection, and our
understanding of the transmission, pathogenesis and phylogeny of bacteria.

In humans (DNA profiling): (See DNA technology IV)

 Used in forensic science
 Paternity and identification studies
 For estimating predisposition to disease (differences in our susceptibility to, or
protection from all kinds of diseases. The severity of illness and the way our body
responds to treatments are also manifestations of genetic variations)
 For mapping and genome-wide association studies of complex diseases
 To predict specific genetic traits

In micro-organisms (genomic typing):

 In epidemiological studies (to identify an organism causing a disease outbreak)
 To determine transmission routes
 For surveillance
 To determine differences in clinical outcome - The identification of pathogenic
factors can help to identify what is different between strains causing and not
causing disease.

Genetic base for typing?

Laboratory techniques such as culture, microscopy, serology, phage-typing, and molecular

methods can be used both to verify the presence of an organism and to identify that
organism. Molecular techniques involving DNA or RNA are particularly powerful tools for

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typing. The useful thing about DNA sequences is that it can be used to identify an organism
causing a disease outbreak. Certain DNA sequences are unique to each organism.

By examination of the right sections of DNA molecules, two different strains of the same
species of microbe can be distinguished from each other. Some sequences will be exactly the
same among strains of the same species, while other sequences will have areas of variation
that can be used to distinguish one strain from another. This property of DNA sequences is
useful in determining whether different cases of the same disease are actually part of an
outbreak.

For example, if Norovirus is identified in two cases of gastrointestinal illness, they may or may
not be part of the same outbreak. If you determine that these are different strains of
Norovirus, you know the cases are not related. If the cases have the same strain, they might
have acquired the infection from the same source, or one case might have transmitted the
infection to the other.

Using molecular techniques such as polymerase chain reaction (PCR) to examine DNA
sequences can help to identify what strain of a pathogen is present in a specimen. If you are
still unsure what the infecting organism might be after performing PCR on the specimen, you
probably ran a non-specific PCR reaction (that is, you amplified whatever genetic material was
present). With the supply of genetic material obtained after amplification, your next step is
to sequence the DNA.

The DNA obtained through PCR can also be further processed to identify its DNA fingerprint,
a pattern on a gel that will identify the organism. DNA fingerprinting is generally done when
a specific organism is suspected, in order to determine which strain of the organism is
present.

For example, tuberculosis (TB) has clear symptoms, but DNA fingerprinting might be used to
determine whether different cases of TB are infected with the same strain, possibly due to an
outbreak or to a common exposure.

Advances in molecular genetics have facilitated the description of the genetic diversity of
bacterial populations. Molecular genetic techniques have been used to distinguish if there
have been independent spontaneous mutations leading to antibiotic resistance or if
resistance was transmitted between strains via a mobile genetic element. In other
applications molecular genetic techniques have determined the flow of infection from one
group to another. These descriptive molecular epidemiologic studies often use strains
collected from disparate areas and the epidemiologic and clinical information is minimal or
non-contributory. In this case the chosen bacterial typing technique must be interpretable in
terms of genetic distance (phylogeny) for the given time period and organism.

SEE: Table 1: Different typing techniques

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Table 1: Different typing techniques

Discriminatory
Technique Time Cost
Power
Sequencing of entire genome High Months to years Very high
Direct sequencing of one or Medium to high
High 2-3 days
more genes only (Equipment)
Micro-arrays High 2-3 days High
Multilocus sequence typing High (Equipment,
Moderate to high >3 days
(MLST) labour)
Pulsed-field gel High (Equipment,
Moderate to high 3 days
electrophoresis (PFGE) labour)

Restriction fragment length

Moderate to high 1-3 days Medium
polymorphism (RFLP)

Random primers (randomly

amplified polymorphic DNA
Low to Moderate 1 day Medium to low
(RAPD), arbitrary primed PCR
(AP-PCR))
Ribotyping Moderate 1 day High
Targeting known repetitive
Low to Moderate 1 day Medium to low
gene sequences
Restriction endonuclease on
Low to Moderate 1-2 days Medium to low
a single amplified product
Plasmid profiles Low
1 day Low

TYPING METHODS
Traditional typing systems for discriminating between bacteria from a single species have
been based on phenotype, such as serotype, biotype, phage typing, or antibiogram
(susceptibility to one or more antibiotics). More recently, techniques have been developed
based on indirect measures of genetic sequence (such as pulsed-field gel electrophoresis
(PFGE)) and direct measures of genetic sequence (such as multilocus sequence typing
(MLST)). Sequencing an entire bacterial genome, and, using microarray technologies,
comparing strains to a reference strain (comparative genomic hybridization) is technically
feasible; however, the cost and time required limits the applicability for most epidemiologic
studies.

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Common typing techniques used in epidemiologic studies sequence one or more genetic
regions, for example multi-locus sequence typing (MLST) and ribotyping, or use enzymes to
cut part or all of the genome into pieces, for example, pulsed-field gel electrophoresis. The
number and size of the pieces correspond to the number and location of restriction sites cut
by the enzymes, and thus are an indirect measure of sequence. Other common techniques
use the polymerase chain reaction targeted random sequences, for example RAPD; the
resulting reactions yield fragments of different sizes, which can be used to discriminate
between bacterial types. Generally speaking, sequence-based methods are most repeatable
and reproducible. Gel-based methods are less so, because of the inherent variability of the
technique.

1. Pulsed Field Gel Electrophoresis (PFGE)

DNA can also be detected by pulsed field gel electrophoresis (PFGE), which is used for the
analysis of large DNA fragments. PFGE is advantageous because it requires less processing
and sample preparation of the DNA. To perform PFGE, special enzymes can be used to cut
the DNA into a few rather long pieces. Instead of applying an electrical field such that the
DNA fragments race straight to the end, after the electrical field is applied, the direction is
changed, and then changed back, and then changed again.

2. Restriction fragment length polymorphism (RFLP)

This technique is usually coupled with PFGE.

The genetic variability at a particular locus (gene) due to even minor base changes can alter
the pattern of restriction enzyme digestion fragments that can be generated. Pathogenic
alterations to the genotypic can be due to deletions or insertions within the gene being
analyzed or even single nucleotide substitutions that can create or delete a restriction
enzyme recognition site. RFLP analysis takes advantage of this and utilizes Southern blotting
of restriction enzyme digested genomic DNA to detect familial patterns of the fragments of
a given gene, detectable by screening the Southern blot with a probe corresponding to the
gene of interest.

i. A classic example of a disease detectable by RFLP is sickle cell anemia:

Sickle cell anemia results (at the level of the gene) from a single nucleotide change (A
--> T) at codon 6 within the -globin gene. This alteration leads to a glu ----> val amino
acid substitution, while at the same time abolishing a MstII restriction site. As a result
a -globin gene probe can be used to detect the different MstII restriction fragments.

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 It should be recalled that there are two copies of each gene in all human cells,
therefore, RFLP analysis detects both copies: the affected alelle and the unaffected
allele.

ii. RFLP is often used to create DNA fingerprints to detect and tract bacterial strains
associated with foodborne outbreaks. This is an example of such an experiment where
Salmonella isolates were digested with Xba1. The patterns are compared with each
other and with a database to determine the strains involved in the outbreak.

Size variability in detectable fragments within a family pedigree indicate differences in the
pattern of restriction sites within and around the gene being analyzed. RFLP patterns are
inherited and segregate in Mendelian fashion thus, allowing their use in genotyping such
as in cases of paternity dispute or in criminal investigations.

Another form of DNA polymorphism detectable by classical RFLP mapping results from
the inherited variations in the number of tandemly repeated DNA sequence elements that
are from 2 to 60 bp in length. The number of repeats is also variable from 2 to 40 copies.
These elements are termed variable number tandem repeats (VNTRs). When restriction
enzyme digestion cuts DNA flanking the VNTRs, the lengths of the resultant fragments will
be variable depending upon the number of repeats at a given locus. Many different VNTR
loci have been identified and are extremely useful for DNA fingerprint analysis such as in
forensic and paternity identity cases.

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3. Ribotyping

Ribotyping is similar to RFLP but where any DNA sequence regardless of its source are cut
with RFLP, only DNA coding for rRNA is cut in ribotyping.

Its name is derived from the ribosome, which is part of the cellular machinery that creates
proteins. Ribosomes are found only in cells, so ribotyping is a method of identifying bacteria,
not viruses. Ribotyping requires bacteria to have > 1 ribosomal set of genes, otherwise they
CAN NOT BE ribotyped. A ribosome is composed of RNA that is folded up on itself in a
particular way. This RNA is referred to as “rRNA” for ribosomal RNA. Since living cells in
everything from lizards to people create proteins, the DNA genes that code for rRNA have
much in common, even across vastly different species. Typically, each ribosomal operon
consists of the three genes encoding the structural rRNA molecules, 16S, 23S, and 5S. The
copy numbers, overall ribosomal operon sizes, nucleotide sequences, and secondary
structures of the three rRNA genes are highly conserved within a bacterial species due to their
fundamental role in polypeptide synthesis. Because the 16S rRNA gene is the most conserved
of the three rRNA genes, 16S rRNA gene sequencing has been established as the “gold
standard” for identification and taxonomic classification of bacterial species.

However, some parts of the genes that code for rRNA are highly variable. That is, certain
sequences are quite different from one species to the next, or even from one strain of bacteria
to the next. These variable regions can be used to identify a particular strain type of bacteria.

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 The variable regions are determined by using restriction enzymes
(as in RFLP). These enzymes cut the RNA only when a specific
sequence occurs. Thus, if a strain of bacteria has that sequence in
its rRNA, the rRNA will be cut at that location. If another strain of
bacteria has a few different bases in the same spot, the rRNA will
not be cut. The rRNA is then run on a gel so the number and size of
the pieces can be seen. rRNA that has been cut in the expected
locations will appear different from rRNA that was not cut.

The advantage of ribotyping as a method of identifying infectious

microorganisms is that automated machines available, and there
are databases with large numbers of reference ribotype patterns
for comparison; software is available to determine relationships
between strains.

Not only is less labor involved in performing the procedure, but the
procedure is standardized. However, because of the equipment
needed, ribotyping is rather expensive, and is usually only
performed in reference laboratories.

4. Multilocus sequence typing (MLST)

Multilocus sequence typing (MLST) is used for the typing of multiple loci. The procedure
characterizes microbial strains using the DNA sequences of internal fragments of multiple
housekeeping genes. Approximately 450-500 bp internal fragments of each gene are used, as
these can be accurately sequenced on both strands using an automated DNA sequencer. For
each housekeeping gene, the different sequences present within a bacterial species are
assigned as distinct alleles and, for each isolate, the alleles at each of the loci define the allelic
profile or sequence type (ST).

The first MLST scheme to be developed was for Neisseria meningitidis

MLST directly measures the DNA sequence variations in a set of housekeeping genes and
characterizes strains by their unique allelic profiles. The principle of MLST is simple: the
technique involves PCR amplification followed by DNA sequencing. Nucleotide differences
between strains can be checked at a variable number of genes depending on the degree of
discrimination desired.

In data analysis all unique sequences are assigned allele numbers and combined into an allelic
profile and assigned a sequence type (ST). If new alleles and STs are found, they are stored in

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database after verification. In the final section of MLST the relatedness of isolates are made
by comparing allelic profiles.

To strike the balance between the acceptable identification power, time and cost for the
strain typing, about seven to eight house-keeping genes are commonly used in the
laboratories. Eg. for Staphylococcus aureus 7 housekeeping genes are used and for Vibrio
vulnificus, 10 genes are used in MLST typing.

For each of these housekeeping genes, the different sequences are assigned as alleles and the
alleles at the loci provide an allelic profile. A series of profiles can then be the identification
marker for strain typing. Sequences that differ at even a single nucleotide are assigned as
different alleles. The relatedness of isolates is displayed as a dendrogram.

Different organisms’ ST types are in a database, and you can compare yours with it.
www.mlst.net/

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5. Random primers (randomly amplified polymorphic DNA (RAPD),
arbitrary primed PCR (AP-PCR))

Arbitrarily Primed PCR (AP-PCR) or Random Amplified Polymorphic DNA (RAPD) are
methods of creating genomic fingerprints from species of which little is known about target
sequence to be amplified.

Strain-specific arrays of DNA fragments (fingerprints) are generated by PCR amplification

using arbitrary oligonucleotides to prime DNA synthesis from genomic sites which they
fortuitously match or almost match.

Unlike traditional PCR analysis, RAPD does not require any specific knowledge of the DNA
sequence of the target organism: the identical 10-mer primers will or will not amplify a
segment of DNA, depending on positions that are complementary to the primers' sequence.
For example, no fragment is produced if primers annealed too far apart or 3' ends of the
primers are not facing each other. Therefore, if a mutation has occurred in the template DNA
at the site that was previously complementary to the primer, a PCR product will not be
produced, resulting in a different pattern of amplified DNA segments on the gel.

DNA amplified is this manner can be used to determine the relatedness of species = DNA
fingerprinting.

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6. Plasmid profile

Plasmids are extra-chromosomal molecules of deoxyribonucleic acid (DNA) capable of

autonomous replication. Such molecules have been identified in many bacterial genera and
usually exist as covalently closed circular (CCC) molecules. Plasmids range in size from less
than one megaDalton (MDa) to several hundred MDa. One megaDalton of double-stranded
DNA is equivalent to 1500 bp or 1.5 kilobases. Many plasmids code for properties such as
antimicrobial resistance, production of toxins or siderophores – virulence factors. Other
plasmids may not have any obvious phenotypic properties and are seen as ‘cryptic plasmids’.

The identification of plasmids (especially the number and molecular weight) by agarose gel
electrophoresis gives a profile for every strain.

Plasmid analysis is a useful method for epidemiologic typing of a variety of organisms. In

outbreaks, plasmid profiling is a rapid, convenient way to follow the spread of the epidemic
strain and may be more specific than other typing methods. Restriction endonuclease
profiling of plasmids has led to an understanding of how the transmission of specific
resistance plasmids among many species can contribute to endemic antibiotic resistance.

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7. High Resolution Melt (HRM) analysis

High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the
detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA
samples. It was discovered and developed by Idaho Technology and the University of Utah. It
has advantages over other genotyping technologies, namely:

 It is cost effective vs. other genotyping technologies such as sequencing and Taqman
SNP typing. This makes it ideal for large scale genotyping projects.
 It is fast and powerful thus able to accurately genotype many samples rapidly.
 It is simple. With a good quality HRM assay, powerful genotyping can be performed
by non-geneticists in any laboratory with access to an HRM capable real-time PCR
machine.

Method

HRM analysis is performed on double stranded DNA samples. Typically the user will use
polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which
their mutation of interest lies. In the sample tube there are now many copies of the DNA
region of interest. This region that is amplified is known as the amplicon. After the PCR
process the HRM analysis begins. The process is simply a precise warming of the amplicon
DNA from around 50˚C up to around 95˚C. At some point during this process, the melting
temperature of the amplicon is reached and the two strands of DNA separate or “melt” apart.

The secret of HRM is to monitor this process happening in real-time. This is achieved by using
a fluorescent dye. The dyes that are used for HRM are known as intercalating dyes and have
a unique property. They bind specifically to double-stranded DNA and when they are bound
they fluoresce brightly. In the absence of double stranded DNA they have nothing to bind to
and they only fluoresce at a low level. At the beginning of the HRM analysis there is a high
level of fluorescence in the sample because of the billions of copies of the amplicon. But as
the sample is heated up and the two strands of the DNA melt apart, presence of double
stranded DNA decreases and thus fluorescence is reduced. The HRM machine has a camera

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that watches this process by measuring the fluorescence. The machine then simply plots this
data as a graph known as a melt curve, showing the level of fluorescence vs the temperature:

Spot the difference

The melting temperature of the amplicon at which the two DNA strands come apart is entirely
predictable. It is dependent on the sequence of the DNA bases. If you are comparing two
samples from two different people, they should give exactly the same shaped melt curve.
However if one person has a mutation in the DNA region you have amplified, then this will
alter the temperature at which the DNA strands melt apart. So now the two melt curves
appear different. The difference may only be tiny, perhaps a fraction of a degree, but because
the HRM machine has the ability to monitor this process in “high resolution”, it is possible to
accurately document these changes and therefore identify if a mutation is present or not.

Wild type, heterozygote or homozygote?

Things become slightly more complicated than this because organisms contain two (or more)
copies of each gene, known as the two alleles. So, if a sample is taken from a patient and
amplified using PCR both copies of the region of DNA (alleles) of interest are amplified. So if
we are looking for mutation there are now three possibilities:

1. Neither allele contains a mutation

2. One or other allele contains a mutation
3. Both alleles contain a mutation.

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These three scenarios are known as “Wild –type”, “Heterozygote” or “Homozygote”
respectively. Each gives a melt curve that is slightly different. With a high quality HRM assay
it is possible to distinguish between all three of these scenarios.

Applications of HRM

SNP typing/Point mutation detection

Conventional SNP typing methods are typically time consuming and expensive, requiring
several probe based assays to be multiplexed together or the use of DNA microarrays. HRM
is more cost effective and reduces the need to design multiple pairs of primers and the need
to purchase expensive probes. The HRM method has been successfully used to detect a single
G to A substitution in the gene Vssc (Voltage Sensitive Sodium Channel) which confers
resistance to the acaricide permethrin in Scabies mite. This mutation results in a coding
change in the protein (G1535D). The analysis of scabies mites collected from suspected
permethrin susceptible and tolerant populations by HRM showed distinct melting profiles.
The amplicons from the sensitive mites were observed to have a higher melting temperature
relative to the tolerant mites, as expected from the higher thermostability of the GC base pair
[2]

In a field more relevant to clinical diagnostics, HRM has been shown to be suitable in principle
for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2.
More than 400 mutations have been identified in these genes.
The sequencing of genes is the gold standard for identifying mutations. Sequencing is time
consuming and labour intensive and is often preceded by techniques used to identify
heteroduplex DNA, which then further amplify these issues. HRM offers a faster and more
convenient closed-tube method of assessing the presence of mutations and gives a result
which can be further investigated if it is of interest. In a study carried out by Scott et al. in

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2006, 3 cell lines harbouring different BRCA mutations were used to assess the HRM
methodology. It was found that the melting profiles of the resulting PCR products could be
used to distinguish the presence or absence of a mutation in the amplicon. Similarly in 2007
Krypuy et al. showed that the careful design of HRM assays (with regards to primer
placement) could be successfully employed to detect mutations in the TP53 gene, which
encodes the tumour suppressor protein p53 in clinical samples of breast and ovarian cancer.
Both these studies highlighted that fact that changes in the melting profile can be in the form
of a shift in the melting temperature or an obvious difference in the shape of the melt curve.
Both of these parameters are a function of the amplicon sequence. The consensus is that HRM
is a cost efficient method that can be employed as an initial screen for samples suspected of
harbouring polymorphisms or mutations. This would reduce the number of samples which
need to be investigated further using more conventional methods.

Zygosity testing

Currently there are many methods used to determine the zygosity status of a gene at a
particular locus. These methods include the use of PCR with specifically designed probes to
detect the variants of the genes (SNP typing is the simplest case). In cases where longer
stretches of variation is implicated, post PCR analysis of the amplicons may be required.
Changes in enzyme restriction, electrophoretic and chromatographic profiles can be
measured. These methods are usually more time consuming and increase the risk of amplicon
contamination in the laboratory, due to the need to work with high concentrations of
amplicons in the lab post-PCR. The use of HRM reduces the time required for analysis and the
risk of contamination. HRM is a more cost effective solution and the high resolution element
not only allows the determination of homo and heterozygosity, it also resolves information
about the type of homo and heterozygosity, with different gene variants giving rise to
differing melt curve shapes. A study by Gundry et al. 2003, showed that fluorescent labelling
of one primer (in the pair) has been shown to be favourable over using an intercalating dye
such as SYBR green I. However, progress has been made in the development and use of
improved intercalating dyes which reduce the issue of PCR inhibition and concerns over non-
saturating intercalation of the dye.

Epigenetics

The HRM methodology has also been exploited to provide a reliable analysis of the
methylation status of DNA. This is of significance since changes to the methylation status of
tumour suppressor genes, genes that regulate apoptosis and DNA repair, are characteristics
of cancers and also have implications for responses to chemotherapy. For example, cancer
patients can be more sensitive to treatment with DNA alkylating agents if the promoter of the
DNA repair gene MGMT of the patient is methylated. In a study which tested the methylation
status of the MGMT promoter on 19 colorectal samples, 8 samples were found to be
methylated.

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Methylated DNA can be treated by bi-sulphite modification, which converts non-methylated
cytosines to uracil. Therefore, PCR products resulting from a template that was originally
unmethylated will have a lower melting point than those derived from a methylated
template. HRM also offers the possibility of determining the proportion of methylation in a
given sample, by comparing it to a standard curve which is generated by mixing different
ratios of methylated and non-methylated DNA together. This can offer information regarding
the degree of methylation that a tumour may have and thus give an indication of the
character of the tumour and how far it deviates from what is “normal”.

HRM also is practically advantageous for use in diagnostics, due to its capacity to be adapted
to high throughput screening testing, and again it minimises the possibility of amplicon spread
and contamination within a laboratory, owing to its closed-tube format.

Intercalating dyes[

To follow the transition of dsDNA (double-stranded) to ssDNA (single-stranded), intercalating

dyes are employed. These dyes show differential fluorescence emission dependent on their
association with double-stranded or single-stranded DNA. SYBR Green I is a first generation
dye for HRM. It fluoresces when intercalated into dsDNA and not ssDNA. Because it may
inhibit PCR at high concentrations, it is used at sub-saturating concentrations. Recently, some
researchers have discouraged the use of SYBR Green I for HRM, claiming that substantial
protocol modifications are required. This is because it is suggested that the lack of accuracy
may result from “dye jumping”, where dye from a melted duplex may get reincorporated into
regions of dsDNA which had not yet melted. New saturating dyes such as LC Green and LC
Green Plus, ResoLight, EvaGreen, Chromofy and SYTO 9 are available on the market and have
been used successfully for HRM. However, some groups have successfully used SYBR Green I
for HRM with the Corbett Rotorgene instruments and advocate the use of SYBR Green I for
HRM applications.

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