POLIMORFISM DNA

Prof. Dr. Sismindari

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 DNA Polymorphisms
What types of DNA polymorphism exist?
RFLP: Restriction fragment-length
polymorphism
SNP: Single nucleotide polymorphism
VNTR: Variable number of tandem repeats
minisatellite
STR: Short tandem repeats
microsatellites
Although there are many variations in
methodology, the basic principal for detection
of DNA variability is differences in the size of
fragments
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 Restriction fragment-length
polymorphism (RFLP)
This was historically the first type of DNA polymorphism
used for genetic mapping
In this type of polymorphism, a change in sequence
causes a restriction endonuclease site to be formed
or to disappear (arises by point mutation)
In most cases, these are diallelic markers, and are
thus of limited utility for general mapping
RFLPs were detected historically after enzymatic
digestion by hybridization with a labelled probe
Currently: PCR, followed by enzymatic digestion and
gel electrophoresis
RFLP
SNP
VNTR
STR
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Genotyping RFLPs

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 Single nucleotide polymorphism
(SNP)

Also a change in a single base pair, but does not

necessarily cause a change in a restriction enzyme site
Biallelic, and equivalent to a point mutation
As compared to RFLPs, SNPs are known to be widely
distributed across the genome, occurring approximately
once in every 500-1000 bp
Given a genome size of 3300 Mb, there must be 4-5
million SNPs in the human genome
SNPs are identified by sequencing, allele-specific
hybridization or allele-specific primer extension
RFLP
SNP
VNTR
STR 5
SNPs and sequence analysis

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A general method for SNP genotyping

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Allele specific amplification

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 Variable number of tandem
repeats (VNTR)

A short sequence of DNA (20-50 nucleotides) repeated a

variable number of times
Multiallelic, and thus highly informative for mapping
VNTRs arise primarily from unequal crossing-over
Not uniformly distributed through the genome:
concentrated at telomeres and in hypervariable regions
Detection is by PCR and electrophoresis, or blotting, or
by (infrequently) by column electrophoresis

RFLP
SNP
VNTR
STR
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VNTR

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Unequal crossing-over

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Complex (fingerprinting) VNTR

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 STR: Short tandem repeats

A very short sequence of DNA (2-6 nucleotides)

repeated a variable number of times
Multiallelic, and thus highly informative for mapping
Typically arise through slipped strand mispairing
Widely distributed, and thus the standard for general
mapping experiments at the present time
Detection is by PCR and electrophoresis (gel or
column), often with automated reading of fluorescent
labels

RFLP
SNP
VNTR
STR
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Segregation of an STR

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Slipped-strand mispairing

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Two other types of DNA site which
have been important in developing
the Human Genome Project

Expressed sequence tag (EST): A nucleotide sequence

which identifies an expressed gene: identified from
cDNA library: not necessarily polymorphic
Sequence-tagged site (STS): A sequenced fragment of
DNA which has been named and located, and for which
a PCR assay has been developed: not necessarily
polymorphic or expressed

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What is the relationship between
DNA polymorphisms and
genetic markers?
A genetic marker is any character which can be traced through
families
Thus, all germ-line polymorphic DNA markers are genetic markers,
but not all genetic markers are (as yet) DNA-based
DNA may also undergo somatic alteration, such that polymorphism
which occurs in somatic tissue may not always be inherited:
example--tumors
The great advantage of DNA polymorphisms is that they can be
scored easily and inexpensively with small amounts of human
material

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 What is the relationship
between variation in DNA and
variation in protein?
When the starting point is a protein mutation or polymorphism,
there is generally a 1:1 relationship with a DNA sequence change
Example: Hb A/S
However, if the starting point is a change in DNA sequence, there
is not necessarily any effect on protein sequence
The DNA sequence change may occur in an untranslated
region
Even in a translated region, the sequence change may produce
a synonomous codon
<10% of changes in protein sequence alter protein function

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Uses of DNA polymorphisms

Mapping
Identification of individuals
Forensics
Better understanding of the mechanisms of
genetic disease (eg., triplet repeats, micro-
deletions or rearrangements)

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TRANSMISSION PATTERN OF
TUBERCULOSIS USING RFLP-
BASED IS6110

Farnia, P., Mohammadi, F., Fadda, G., Sanguinetti, M.,

Ghazisaeed, K., Tabatabaei, S.J., Posteraro, B.,
Mansoori, D., Bahadori, M., Masjedi, M.R., Velayati,
A.A.
Arch. Irn. Med. 2001;4(4):177-182

Sophie Kulaga, Marchel A. Behr, Kevin Schwartzman

CMAJ. Nov.2, 1999; 161(9)

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 Keterbatasan Penggunaan RFLP DNA
Fingerprinting dalam Pola Penularan TB
Sebagian besar isolat MTB mengandung multiple copies
IS6110, tetapi ada beberapa isolat yang hanya memiliki
very few copies atau bahkan tidak ada
Suatu metode yg didasarkan pada PCR-spacer
oligonucleotide typing (spoligotyping) telah dikembangkan
untuk identifikasi cepat MTB dan sedang dievaluasi
kegunaan utk deferensiasi isolat dengan sedikit/ tidak ada
kopi IS6110
Genetic fingerprinting hanya dapat digunakan pada kasus
aktif TB, tetapi tdk dapat digunakan utk identifikasi orang
dengan latent infection (sekitar 90% dari populasi yg
terinfeksi), termasuk resiko tinggi terjadi reaktifasi dimasa
datang 25
 Deteksi mutasi M.tuberculosis
resistensi obat

Hajj et al, 2001, Detection of rifampin resistance in Mtuberculosis in a single tube with
Molecular beacons, J.Clin.Microbiol., 39: 4131-4137

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Molecular beacon

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 RAPD, or Arbitrary-primed
PCR

Isolate DNA
PCR using a short
(8-12 base) primer
of arbitrary
sequence
Electrophorese
and visualize 31
 RAPD example
Muyldermans, G., Bougatef, A., Naessens, A. and Lauwers, S. (2001) Outbreak
of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered
milk formula. J Clin Microbiol, 39, 293-7.
RAPD using enterobacterial repetitive intergenic consensus
motif primers
14 isolates from powdered milk were identical to isolates from 3/6
sick babies
RAPD is good for quick and dirty answers in an outbreak
investigation

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Random Amplified Polymorphic DNA (RAPD)
Fingerprinting
In RAPD, arbitrary oligonucleotide primers are used in
PCR.
As a result, random banding patterns are produced.
These patterns (fingerprints) can be used as an easily
obtainable typing scheme.
It is used as a tool to differentiate between genospecies
and strains.
RAPD can also be used as a tool to study the change in
the genetic make up of bacteria existing at different
places and time.

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RAPD fingerprints of a 1929 isolate (CH31) and some curent isolates

LaneM: Marker 100bp ladder

Lane 1: CH 31 (1929 isolate)
Lane 2: D 22
Lane 3: Mg 47
Current
Lane 4: Mg 51
isolates
Lane 5: Mg 100.

Primer PB1 Primer L10 Primer M16 (S6)

These pictures also show that the M16 primer produced better and clearer bands
than the other 2 commonly used primers.

Roy et.al., J. Med.Microbiol.52. 2003

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Evaluation of discriminatory power of M16 (S6) primer
RAPD fingerprints of the reference strains representing different seogroups/serovars

Fingerprints obtained were all different from each other

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RAPD fingerprints of 10 reference strains belonging to different
serovars of Grippotyphosa with dfferent primers.

Primer PB1 Primer L10

Primer M16

Primers PB1 & L10 cannot differentiate between some of these reference
strains but primer M16 can differentiate between all.
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 RAPD fingerprints of 2 strains of the same serovar

Lane 1: Grippo Moskva V (Grippotyphosa)

Lane 2: CH31 (Grippotyphosa)

Primer M16 was able to differentiate between 2 strains of the same serovars
of the serogroup.

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RAPD fingerprints of 24 isolates with M16 primer

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 CONCLUSIONS

Most isolates can be typed by RAPD. In this study, 23 out of

24 isolates could be typed as genetically identical with a
certain type of reference strain by this method.
Occasionally some isolates cannot be matched genetically to
any of the reference strains used. In this study, 1 isolate was
found to belong to Sejroe serogroup but genetically could not
be matched to any reference strain. Though antigenically
similar, it could be genetically different from the reference
Sejroe Hardjo prajitno strain used here.

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 CONCLUSIONS (Contd)

Often, the genetic and serological characterizations do

not match. In this study, 1 isolate was found
genetically similar to Pomona reference strain, but
MAT showed closer relationship to Autumnalis.
Clonality of strains could also be well detected by this
technique. In this study, many isolates produced
identical banding patterns.
The technique is quick and simple but a major
problem with this technique is the reproducibility in
different conditions and different laboratories.

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 GLUTAMAT VALIN
Kodon glutamat : GUU, GUC, GUA, GUG
Kodon valin : GAA, GAG
Pengenalan CvnI :
Pengenalan MstII: -CCTNAGG

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PCR-RFLP

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Mana yang:
sehat ?
penderita ?

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 Conformation-dependent
analyses
Single Strand Conformational
Polymorphism (SSCP)
PCR to generate DNA of interest
Denature DNA with heat
Electrophorese and visualize
Otherwise-identical ssDNA
molecules that differ by only a
single nucleotide (or more) will
form secondary structures that
cause them to migrate different
distances on gels 50
 Conformation-dependent analyses

Denaturing gradient gel

electrophoresis (DGGE)
Similar to SSCP, except that double-
stranded DNA is denatured by a
gradient of chemicals (formamide or
urea) in the gel

Gradient
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 Conformation-dependent analyses

Problem: these are difficult to perform

Problem: if two bands differ, this technique does
not tell us the magnitude of the difference
DGGE and similar techniques have very limited
application to molecular epidemiology
Wouldnt it be nice to know the exact sequence of
DNA in a fragment/PCR product?

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 MUATION ANALYSIS in
DUCHENNE/BECKER
MUSCULAR DISTROPHY

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Variation exploited by Forensic
DNA analysis

polymorphism = many variants

Sequence polymorphism occurring

in the sequence of base pairs at a
particular locus
Length polymorphism variation in
length at a particular locus

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 Satellite DNA
A. Repetitive DNA
1. In two major classes:
a. Tandemly repetitive sequences of satellite DNA (about
10% of the genome).
b. Interspersed repetitive DNA makes up 520% of the
genome. They are scattered throughout the genome and
are further subdivided:
1) SINES (short interspersed elements) are sequences of
fewer than 500 bp.
2) LINES (long interspersed elements) are sequences of 500
bp or more.
2. Found near the centromere of chromosomes and also scattered
about the genome. Only found in eukaryotic organisms, without
a known biological function.

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 Satellite DNA Continued
3. Variable Number of Tandem Repeats (VNTRs).
a. Made of a short sequence, or motif, called a core
sequence, which is repeated many times in a row.
b. Inherited from the parents.
c. Core sequences range from two to six bp to over 300 bp
long.
d. The number of times the core sequence is repeated is
variable.
e. A VNTR refers to a single location with different alleles,
characterized by differences in the number of times the
core sequence is repeated.
f. Can have hypervariable loci where many different alleles
exist.
g. Jeffreys was able to use DNA probes for VNTRs by using
Southern blotting to yield a DNA fingerprint.
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h. Can generate by restriction enzyme digestion or PCR.
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 Satellite DNA Continued
3. The number of alleles can also be very high, which means that
it is hypervariable.
4. Loci may number in the thousands, and the sequences are
used to detect length polymorphisms scattered throughout the
genome (called multilocus profiles).
D. Macrosatellites
1. Located near the centromeres and telomeres.
2. Megabases in length, and need a special type of
electrophoresis called pulsed-field gel electrophoresis.
3. Length makes them subject to breakage, so they are not used
in forensic analysis.

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 Population Genetics and Alleles
A. Because humans are diploid organisms, each individual
has two alleles per locus.
B. Individuals could be:
1. Homozygoustwo copies of the same overall length,
even though the DNA sequence may be different.
2. Heterozygoustwo copies of different overall length.
C. Many alleles exist in a population with the maximum
number of alleles being two times the number of people in
the population.
D. Some DNA regions can be hypervariable (usually used in
forensic analysis), while others are not as variable.
E. Allelic polymorphisms in a population are maintained by
genetic drift or natural selection.
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 Multilocus Minisatellite VNTR
A. Supported by genetic and population data, and the data provided by
the probes are reliable and are used in paternity and immigration
cases.
B. More difficult to interpret than single-locus patterns for several reasons:
1. The large number of bonds usually generated.
2. Incomplete cutting of the DNA.
3. DNA degradation.
4. Low DNA recovery.
5. Identification problems with mixed DNA samples from more than
one individual.
C. DNA fingerprinting has still been successful in forensic cases and
evaluating the genetic diversity of plants and animals.
D. Usually detected using Southern blot hybridization.

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 Single-Locus VNTR for STR
A. Generate only one DNA fragment (homozygous) or two fragments
(heterozygous).
B. If more than one location is used, the more informative the analysis.
C. Is technically less difficult because it eliminates the possibility of co-migration of
alleles, and it is less likely to produce artifacts.
D. The method of choice today is using PCR, by using at least four or five single-
locus primer sets in separate PCR reactions or in the same reaction (called
multiplexing).
E. Some potential problems with multiplexing that need to be overcome:
1. Primers from one locus can sometimes complex with those of other loci.
2. One locus may not amplify as well as another locus.
3. The optimization of PCR conditions can present a challengeannealing
temperature of primers and primer concentrations must be determined and
a uniform annealing temperature may be difficult to determine.
4. If the problems are not resolved, the absence of a specific STR allele may
not actually represent the individuals genotype. An alternative may be to
recover the DNA from one reaction, and use it in another reaction (called
sequential multiplex amplification).
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 Genetic polymorphisms of
drug-metabolising enzymes, transporters, receptors or
targets

PHASE I ENZYMES

GENE REPRESENTATIVE DRUG CONSEQUENCES FOR

DRUG EFFECTS
CYP2C9 WARFARIN Anticoagulant effect
CYP2C19 OMEPRAZOLE Peptic ulcer cure rates
CYP2D6 IMIPRAMINE Dose requirement
CYP2D6 CODEINE Narcotic side effects
NQO1 MENADIONE Urolitiasis

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 Genetic polymorphisms of
drug-metabolising enzymes, transporters, receptors or
targets

PHASE II ENZYMES

GENE REPRESENTATIVE DRUG CONSEQUENCES FOR

DRUG EFFECTS

NAT2 ISONIAZID Neurotoxicity

TPMT MERCAPTOPURINE, Toxicity, efficacy
THIOGUANINE
UGT IRINOTECAN Glucuronidation
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 Genetic polymorphisms of
drug-metabolising enzymes, transporters, receptors or
targets

TRANSPORTERS

GENE REPRESENTATIVE DRUG CONSEQUENCES FOR

DRUG EFFECTS

MDR-1 DIGOXIN High plasma levels

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 Genetic polymorphisms of
drug-metabolising enzymes, transporters,
receptors or targets

RECEPTORS/TARGETS

GENE REPRESENTATIVE DRUG CONSEQUENCES FOR

DRUG EFFECTS
2-ADREN. ALBUTEROL Asthma response
CETP PRAVASTATIN Atherosclerosis resp.
SEROTONIN FLUVOXAMINE Antidepressant resp.
D2 & D3 ANTIPSYCHOTICS Dyskinesia
APOE4 TACRINE Alzheimer resp.
RYANODINE HALOTHANE Malignant hyperthermia
PROTHROMBIN CONTRACEPTIVES Thrombosis
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 POLYMORPHIC GENES IN POPULATIONS
OF DIFFERENT ORIGIN

GENE GENOTYPE CAUCASIANS ASIANS BLACK ETHIOPIANS &

AFRICANS SAUDI
ARABIANS
POOR METABOLIZERS %
CYP2C9 Homozygous PM 0.2-1 ND ND ND

Heterozygous 14-37 2-3 0.5 ND

CYP2D6 Homozygous PM 5-13.5 0-1 0-8 2

NAT2 Homozygous 40-70 9-22 50-60 80

(slow)

ULTRARAPID METABOLIZERS %
CYP2D6 Duplication 1-7 2 2 10-16

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 50% of individuals have DELETIONS -
CYP2D6 not present in human genome
database ! (Cfr. Tryptase II gene on Chr
#16)

CYP2D6
Chromosome 22
Single functional
gene
497 Amino Acids
50 kDa Protein

1 2 3 4 5 6 7 8 9

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Simple Sequence
Length
Polymorphisms
(SSLP) and how
they are typed

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 A single Nucleotide Polymorphism
(SNP)

Allele 1

..AGTCAGAAATC...

Allele 2
..AGTCACAAATC...

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 Pharmacogenomics and Drug
Responsiveness

Polymorphism in genes is common

Cargill et al., 1999 Nature Genetics 22, 231-238
Surveyed 106 genes
1 SNP per 350 bp 5 SNPs per gene
On average:

- 2 silent SNPs
- 2 conservative SNPs
- 1 major change SNP

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 Pharmacogenomics and Drug
Responsiveness

Documented examples

- ALOX 5 Promoter
- Cholesteryl Ester Transfer Protein
- 5 HT2 A Receptor
- Apolipoprotein E4

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