(Molecular Methods in SNP

detection)

Lec. Mohammed H. Wali

College of Applied Biotechnology
Dep. Molecular & Med. Biotech
 Outline
 Introduction
 SNPs Types
 SNPs website
 Retrieval Process
 SNPedia
 Other tools and software
 Detection Methods
Lec. Mohammed H. Wali
 Genetic Polymorphism

It is a difference in DNA sequence among

individuals, groups, or populations. Sources
include SNPs, sequence repeats, insertions,
deletions and recombination. (e.g. a genetic
polymorphism might give rise to blue eyes
versus brown eyes, or straight hair versus curly
hair).
 Genetic Polymorphism

Genetic polymorphisms may be the

result of chance processes, or may have
been induced by external agents (such as
viruses or radiation). If a difference in
DNA sequence among individuals has
been shown to be associated with
disease, it will usually be called a genetic
mutation.
 Genetic Polymorphism

Changes in DNA sequence which

have been confirmed to be caused by
external agents are also generally called
”mutations” rather than ”polymorphisms.”

Lec. Mohammed H. Wali

 SNP

A DNA sequence variation that

occurs when a single nucleotide (adenine,
thymine, cytosine, or guanine) in the
genome sequence is altered and the
particular alteration is present in at least
1% of the population. Also called single
nucleotide polymorphism.
 SNP Types
1. A transition substitution occurs
between purines (A, G) or between
pyrimidines (C, T). This type of
substitution constitutes two thirds of all
SNPs.
2. A trans-version substitution occurs
between a purine and a pyrimidine.
Lec. Mohammed H. Wali
 SNP VS SNV
SNP SNV
Nucleotide changes detected in at Any nucleotide variations observed
least 1% of the population regardless of frequency
Common among population Detected in a group of study. (de
(patients) novo) or common mutations
Associated with genetic traits, Can be linked to a condition or
specific disease and some disease in specific group
syndromes.
Served as a genetic marker NA
NA SNV can be consider as SNP if the
variations are detected in at least
1% of population
 Coding Region SNPs

A SNP in a coding region may have

two different effects on the resulting
protein: Synonymous the substitution
causes no amino acid change to the
protein it produces. This is also called a
silent mutation. Non-Synonymous the
substitution results in an alteration of the
encoded amino acid.
 Missense Mutation
It changes the protein by causing a
change of codon. A nonsense mutation
results in a misplaced termination codon.
One half of all coding sequence SNPs
result in non-synonymous codon
changes.

Lec. Mohammed H. Wali

 SNPs in regulatory regions
These SNPs are capable of
changing the amount or timing of a
protein‟s production. Such SNPs are
much more difficult to find and understand
and gene regulation itself is not yet clearly
understood.
 SNP

is the abbreviation for “single

nucleotide polymorphism,” db (database)
SNP is a public archive of all short
sequence variation, not just single
nucleotide substitutions that occur
frequently enough in a population to be
termed polymorphic.
 SNP Database
dbSNP includes a broad collection of
simple genetic variations such as
single-base nucleotide substitutions,
small-scale multi-base deletions or
insertions, and microsatellite repeats.

Lec. Mohammed H. Wali

 SNP Database
Data submitted to dbSNP can be from any
organism, from any part of a genome, and can
include genotype and allele frequency data if
those data are available. dbSNP accepts
submissions for all classes of simple sequence
variation, and provides access to variations of
germline or somatic origin that are clinically
significant.
SNP database search engine on the NCBI website
A selected gene of interest can be entered in the search
engine
The below example shows some SNPs in vitamin D receptor
VDR.
Show flanks
• Each SNP has its unique ID number
started with “rs” followed by specific Id
number. It stands for “Reference SNP
cluster ID”
• This unique ID allows a quick online
searching for known SNP on any gene
that is recorded on the NCBI database.
So if the above ID is typed in the SNP
NCBI database or even in the Google
search engine, the same page will show
up.
 • For each SNP there is some information
on the nucleotide variation showing the
sequence where the variation occurs.
For example, in the above figure there
are three possibilities “A / C / G”
depending upon the allele type.

Lec. Mohammed H. Wali

• The other information includes
chromosomal location, gene
abbreviation, functional consequences
in which explaining the region where the
SNP is located; exon, intron, 3 prime-
UTR variant, etc
 3 prime-UTR variant
• A genetic change that takes place in the 3'
untranslated region of a gene, such as an SNP or
insertion/deletion, is referred to as a 3' UTR
(untranslated region) variant. Located downstream
(toward the 3' end) of the protein-coding sequence,
the 3' UTR is a segment of messenger RNA (mRNA)
that is crucial for post-transcriptional control of gene
expression even though it is not translated into a
protein.
• The 5 prime is located upstream of the protein-coding
sequence
• When we click on the ID number
(rs9729) the below page will
appear which includes all
information regarding the selected
SNP as mentioned below.
• When we click on „Genomic View” (see
black arrow), detailed information on
related SNPs in the genome will appear.
See the below figure.
 • In the above window you can see the
location of rs9729 SNP on the VDR
gene sequence. In addition, there a
number of detected SNPs can be seen
around in the same gene sequence.
With each SNP, the nucleotide variation
is recorder. Click on SNR rs number to
find out more about each SNP.

Lec. Mohammed H. Wali

Very easy to work with !
Lec. Mohammed H. Wali
A summary window on each SNP
mRNA variant details
 SNPedia
• SNPedia is a wiki-based bioinformatics
web site that serves as a database of
single nucleotide polymorphisms. Each
article on a SNP provides a short
description, links to scientific articles
and personal genomics web sites, as
well as microarray information about
that SNP
A summary table
 SNP Examples
• rs3825018
• rs475688
• rs 2228570
• rs 731236
• rs 1946518
 There are many other websites and software tools for
targeting SNPs.

 SNP – Sites ( Extracting SNPs from a multi-FASTA alignment)

 SNP- Sties (Software for SNPs identification and loclization)

 miRdSNP (a database of disease-associated SNPs and

microRNA target sites on 3'UTRs of human genes)

 SNPsnap: (a Web-based tool for identification and annotation

of matched SNPs)
Lec. Mohammed H. Wali
Lec. Mohammed H. Wali
 Molecular Methods in SNP detection

1- Polymerase Chain Reaction (PCR)-based Methods

2- DNA Sequencing
3- Microarray-Based Methods
4-High-Resolution Melting (HRM) Analysis
5- Fluorescent Resonance Energy Transfer (FRET)
6- Single Molecule Real-Time (SMRT) Sequencing (PacBio)
7-Digital PCR (dPCR)
8- CRISPR/Cas-Based Methods
9- MALDI-TOF Mass Spectrometry
 Polymerase Chain Reaction (PCR)-based
Methods
• Tradition PCR detection (Allele Specific)
Pros: Fast and simple
Cons: Limited
• Restriction Fragment Length Polymorphism (RFLP) –
Based on Restriction Enzyme (RE)
Pros: Specific
Cons: limited to RE Availability
• TaqMan® Assay (Probe-based PCR) – Real Time PCR
Pros: Very Specific, Multiplex SNP detection and sensitive
Cons: Specific probe design and expensive
 PCR using Tetra Arm Primer
• It uses two primer sets (Outer and Inter)
Outer: the first set amplifies a region
within the SNP sites (wild and mutant)
Inter: the second set designed with a
forward primer anneal to the SNP sites,
while the reverse primer anneal to
adjacent locations near the SNP site
OF + IR=A allele
Tetra-Primer PCR Results
Tetra Arm Primer (ARMS)
and Genotype Illustration
 Designing the Tetra arm Primer

• Primer-BLAST
• Geneious
• Primer3Plus
• Oligo 7
• Benchling (cloud-based platform)
• IDT PrimerQuest
• SnapGene
Restriction Fragment Length Polymorphism
(RFLP)

• Restriction enzymes are proteins

produced by bacteria to prevent or
restrict invasion by foreign DNA
• They act as DNA scissors, cutting the
foreign DNA into pieces so that it cannot
function
 DNA Methyltransferase

Structures of the three primary methylated DNA bases in

prokaryotes and eukaryotes
 Examples of restriction enzymes
• Eco R1: E coli (G|AATTC)
• Haemophilus aegyptius (GG|CC)
• Hae III: Hind III: Haemophilus influenza (A|AGCTT)
• Not I: Norcardia otitidis-caviarum (GC|GGCCGC)
• Eco RV Escherichia coli J62 (GAT|ATC)
• Taq I Thermus aquaticus YT1 (T|CGA)
• Bgl II Bacillus globigii (A|GATCT)
• Sma I Serratio marcescens S (CCC|GGG)
 Type of Cuts by RE

• 5‟-overhang 5 overhang
• 3‟-overhang (protruding)
• Blunt end
Blunt End
Demonstration of allele detection using restriction enzyme
 Choosing the best restriction enzyme

• There are 250 commercially available restriction

enzymes and over 2,000 in total. Selecting a
suitable restriction enzyme depends on:

• Cutting frequency
• Easy to work with
• Cost
TaqMan® Assay
 2- DNA Sequencing
• Sanger Sequencing
- Pros: reliable, accurate, sensitive, specific
- Cons: expensive and time consuming
• Next-Generation Sequencing (NGS)
Illumina, PacBio, Oxford Nanopore
- Pros: High throughput, comprehensive and can
detect rare variants
- Cons: complex data analysis, requires sepcific
software and costly
Snager Sequencing
NGS
 3- Microarray-Based Methods
• SNP microarrays, often referred to as SNP chips, are a high-
throughput method for identifying known SNPs in a large
number of participants in a single study.

a. SNP Genotyping Arrays: An array containing probes unique

to known SNP loci is hybridized to DNA. The hybridization pattern
identifies each allele's existence, and each SNP's genotype is
referred to.

b. Illumina BeadArrays, Affymetrix SNP Arrays: Thousands to

millions of probes that match known SNP locations are present in
these arrays. They function by identifying colorimetric or
fluorescence signals that signify the existence of particular alleles.
Microarray
Beads-based
methods
4-High-Resolution Melting (HRM) Analysis
• HRM is a post-PCR technique that examines DNA
amplicon melting curves. Due to their impact on DNA
structure, SNPs alter the melting temperature of
DNA, and HRM is able to distinguish between alleles
by observing variations in melting behaviour.

• Each SNP may give different melting curve pattern

High-Resolution Melting
 5- Fluorescent Resonance Energy Transfer
(FRET)

• Two fluorescently labeled probes, one for each allele,

are used in FRET-based experiments. When both of
these probes attach to the target sequence,
fluorescence energy is transmitted between them,
creating a signal. These probes are made to
hybridize close to one another.
Fluorescent Resonance Energy Transfer
 6. Single Molecule Real-Time (SMRT) Sequencing
(PacBio)

• Long read lengths are provided by SMRT

sequencing, which is very helpful for identifying
difficult SNPs, such as those with structural variation
or in repetitive sections. It can identify variances
straight from the sequencing data and detect base
changes.
(SMRT) Sequencing (PacBio)
Real Time Sequencing Genotyping
Detection
 7. Digital PCR (dPCR)

• The sample is divided into several separate

reactions using digital PCR, each of which
contains a very small quantity of DNA. The
basis for SNP genotyping is whether or not
particular alleles in these distinct divisions are
amplified.
 8. CRISPR/Cas-Based Methods

• CHRISPR stands for (Clustered Regularly

Interspaced Short Palindromic Repeats)

• This innovative method allows CRISPR/Cas

systems to be engineered to specifically
target particular SNPs. The genotype at the
SNP location can be determined by the
presence or lack of cleavage.
CRISPR Based
Method
 9. MALDI-TOF Mass Spectrometry

• Matrix-assisted laser desorption/ionization

time-of-flight (MALDI-TOF) analyzes the
mass of particular DNA fragments produced
from the PCR product in order to identify
SNPs. Mass spectrometry can detect
changes in the length or sequencing of the
amplicons caused by SNPs.
 Summary of the Detection Methods

Method Detection Type Strengths Limitations

Allele-Specific PCR PCR-based Simple, inexpensive, fast Only detects known SNPs

Limited to SNPs affecting

RFLP PCR-based + Restriction Good for specific loci, simple
restriction sites
Highly specific, sensitive, multiplexing
TaqMan® PCR PCR-based + Probe Costly, requires probe design
possible
Accurate, gold standard for SNP Expensive, low throughput for
Sanger Sequencing Direct sequencing
detection large datasets

High-throughput, detects all SNPs, rare Expensive, complex data

NGS Direct sequencing
variants analysis

High-throughput, cost-effective for Limited to known SNPs, less

Microarrays Hybridization-based
known SNPs sensitive

High-throughput, no need for specific

HRM Analysis PCR-based + Melting curves Less sensitive for rare SNPs
probes
Requires equipment and probe
FRET Hybridization-based Multiplexing possible, highly specific
design
Expensive, less suitable for
SMRT Sequencing Long-read sequencing Detects complex SNPs, high accuracy
high-throughput
Digital PCR PCR-based Very sensitive, quantitative Low throughput, expensive

CRISPR/Cas-based Gene-editing-based High specificity, adaptable to new SNPs Still under development

MALDI-TOF MS Mass spectrometry

Thanks for your attention