MICROARRAY

A microarray is a laboratory tool used to detect the

expression of thousands of genes at the same time
 DNA Microarrays:

• DNA microarrays are microscope slides that are

printed with thousands of tiny spots in defined
positions, with each spot containing a known DNA
sequence or gene. Often, these slides are referred to
as gene chips or DNA chips. The DNA molecules
attached to each slide, act as probes to detect gene
expression, which is also known as the transcriptome
or the set of messenger RNA (mRNA) transcripts,
expressed by a group of genes.
Process:
To perform a microarray analysis, mRNA molecules are typically collected from both an
experimental sample and a reference sample.
For example, the reference sample could be collected from a healthy individual, and
the experimental sample could be collected from an individual with a disease like
cancer.
The two mRNA samples are then converted into complementary DNA (cDNA), and
each sample is labeled with a fluorescent probe of a different color. For instance, the
experimental cDNA sample may be labeled with a red fluorescent dye, whereas the
reference cDNA may be labeled with a green fluorescent dye. The two samples are
then mixed together and allowed to bind to the microarray slide.
The process in which the cDNA molecules bind to the DNA probes on the slide is
called hybridization.
Interpretation of results:
• Following hybridization, the microarray is scanned to measure the expression of each
gene printed on the slide.

• If the expression of a particular gene is higher in the experimental sample than in the
reference sample, then the corresponding spot on the microarray appears red.

• In contrast, if the expression in the experimental sample is lower than in the reference
sample, then the spot appears green.

• Finally, if there is equal expression in the two samples, then the spot appears yellow.

The data gathered through microarrays can be used to create gene expression profiles,
which show simultaneous changes in the expression of many genes in response to a
particular condition or treatment.
Applications of Microarray
 Microbial detection and identification
 Respiratory viral pathogen detection in connection with Multiplex PCR
amplification
 Detection and typing of Human Papilloma Viruses
 Rapid detection and characterization of Methicillin resistant Staph.
Aureus
 Detecting antimicrobial drug resistance
 Microbial gene expression profiling
 Host gene expression profiling during microbial infections
 Detection of chromosome gains/losses and mutations
 Tumor classification and cancer research
 Gene expression profiling and genetic engineering
SEQUENCING
In genetics and biochemistry, sequencing means to determine the primary
structure of an unbranched biopolymer
TYPES OF SEQUENCING
DNA sequencing:
DNA sequencing is the process of determining the nucleotide order of a
given DNA fragment. So far, most DNA sequencing has been performed
using the chain termination method developed by Frederick Sanger. This
technique uses sequence-specific termination of a DNA synthesis reaction
using modified nucleotide substrates
Sanger’s Method:
In chain terminator sequencing (Sanger sequencing), extension is initiated at a
specific site on the template DNA by using a short oligonucleotide 'primer'
complementary to the template at that region. The oligonucleotide primer is
extended using a DNA polymerase, an enzyme that replicates DNA. Included with
the primer and DNA polymerase are the four deoxynucleotide bases (DNA
building blocks), along with a low concentration of a chain terminating nucleotide
most commonly a di-deoxynucleotide
Next Generation Sequencing

Next-generation sequencing (NGS) is a method of DNA sequencing that

allows for the rapid and high-throughput determination of the
nucleotide sequence of a DNA molecule at a much faster rate and lower
cost than traditional Sanger sequencing methods
Principle:
The principle of NGS is based on the ability to synthesize DNA
strands using short, complementary DNA strands known as
sequencing by synthesis.
 Method:
-In NGS, a sample of DNA or RNA is first amplified using polymerase
chain reaction (PCR) or another amplification method. The amplified
DNA or RNA is then fragmented into small pieces and attached to a
solid surface (such as a glass slide or a flow cell).
-Next, the DNA or RNA strands are sequenced using a sequencer.
the sequencer uses short, complementary DNA strands (known as
“sequencing primers”) to synthesize longer DNA strands. As each
nucleotide is added to the synthesized DNA strand, it is detected and
recorded by the sequencer. This process is repeated until the entire
DNA or RNA molecule has been sequenced.
Types of NGS
• Untargeted
• Targeted
Untargeted NGS
-examines all the nucleic acids in a sample

•Whole genome sequencing

•Whole metagenomic sequencing
•Whole transcriptome sequencing
Targeted NGS

relies on primers or probes to target distinct genetic regions of a

sample. Examples of targeted NGS approaches include:

•Targeted methyl sequencing

•Targeted RNA sequencing
•Whole exome sequencing
•Targeted metagenomic sequencing (amplicon based seq)
NGS application

• Rapidly sequence whole genomes

• Cancer research
• Infectious disease research
• Genetic disease research
• Identify novel pathogens
 LIMITATIONS OF NGS:

 Cost: NGS can be expensive, especially for large projects or when multiple
samples need to be analysed.
 Data analysis: NGS generates a large amount of data, which can be difficult
to analyse and interpret.
 Quality of samples: The quality of the samples being analysed can affect
the accuracy and reliability of the results. Poor quality samples can lead to
errors in the data or inaccurate conclusions.
 Depth of coverage: NGS can only provide information about the sequences
that are present in the sample being analysed. If the sample is not
representative of the entire genome or transcriptome, the results may not be
accurate.
 Allele-specific expression: NGS can only provide information about the
overall expression of a gene, rather than the expression of specific alleles
(variants) of a gene.
• Genetic variations: NGS can identify genetic variations, but it may not be
able to determine the functional consequences of those variations
COMPANIES USING NGS TECHNOLOGY

• Illumina
• Agilent technologies
• Bio- Rad lab
• Thermo Fisher Scientific
• Qiagen
• Perkin Elmer
• Roche
Pyrosequencing
Pyrosequencing is a sequencing method that determines the sequence of
nucleotides by detecting the release of pyrophosphate (PPi) during nucleotide
incorporation and monitors DNA synthesis in real-time.

Pyrosequencing relies on light detection which occurs due to a chain reaction

initiated by the release of pyrophosphate
•When released pyrophosphate combines with substrate known as Adenosine
Phosphosulphate (APS) in the presence of an enzyme ATP sulfurylase, The
ATP is generated.
PPi + APS ➞ ATP + Sulfate (catalyzed by ATP-sulfurylase)
Reaction 2
•In the next reaction this ATP is utilized by the enzyme luciferase for the
conversion of Lucifer into Oxyluciferin and production of light.
ATP + luciferin + O₂ ➞ AMP + PPi + oxyluciferin + CO₂ + Light (catalyzed by
luciferase);
Thus the pyrophosphate released during DNA synthesis can be detected by the
emission of light. This detection of pyrophosphate is the basis of DNA
sequencing and hence the name pyrosequencing.
Protein sequencing
Methods for performing protein sequencing include:
Edman degradation
Edman degradation, developed by Pehr Edman, is
a method of sequencing amino acids in a
peptide. In this method, the amino-terminal
residue is labeled and cleaved from the peptide
without disrupting the peptide bonds between
other amino acid residues.
 Peptide mass fingerprinting
 Mass spectrometry
 Protease digests
If the gene encoding the protein is known, it is currently
much easier to sequence the DNA and infer the protein
sequence. Determining part of a protein's amino-
acid sequence (often one end) by one of the above
methods may be sufficient to identify a clone carrying this
gene.
Whole exome sequencing
Exome sequencing, also known as whole exome sequencing (WES), is
a genomic technique for sequencing all of the protein-coding regions of genes in
a genome (known as the exome).
It consists of two steps:
1. select only the subset of DNA that encodes proteins. These regions are
known as exons—humans have about 180,000 exons, constituting about 1%
of the human genome, or approximately 30 million base pairs.
2. The second step is to sequence the exonic DNA using any high-
throughput DNA sequencing technology.
GOAL:
• to identify genetic variants that alter protein sequences
• to do this at a much lower cost than whole-genome sequencing
• Since these variants can be responsible for both Mendelian and
common polygenic diseases, such as Alzheimer's disease, whole exome
sequencing has been applied both in academic research and as a clinical
diagnostic tool.
Whole Genome
sequencing
Whole genome sequencing (WGS) is the process of determining the entirety of
the DNA sequence of an organism's genome at a single time. This entails
sequencing all of an organism's chromosomal DNA as well as DNA contained in
the mitochondria and, for plants, in the chloroplast.
In 1999, the entire DNA sequence of human chromosome 22, the second
shortest human autosome, was published.

In 2008, a group from the Netherlands, reported the sequencing of the first
female human genome (Marjolein Kriek).
Cells used for sequencing:
Almost any biological sample containing a full copy of the DNA e.g.

• Saliva

• epithelial cells

• bone marrow

• hair (as long as the hair contains a hair follicle)

• seeds, plant leaves, or anything else that has DNA-containing cells.

Single cell genome sequencing is being tested as a method of preimplantation

genetic diagnosis, wherein a cell from the embryo created by in vitro fertilization is
taken and analyzed before embryo transfer into the uterus. After implantation, cell-
free fetal DNA can be taken by simple venipuncture from the mother and used for
whole genome sequencing of the fetus.
Comparison with DNA microarray

Full genome sequencing provides information on a genome that is many folds

larger than by DNA arrays.
For humans, DNA arrays currently provide genotypic information on up to one
million genetic variants, while full genome sequencing will provide information on all
six billion bases in the human genome, or 3,000 times more data.

Also it is more cost effective since both have the same accuracy range.
Applications:
 Establishment of mutation frequencies

 Can be used in a Genome-Wide Association Study (GWAS) – a project aiming to determine the
genetic variant or variants associated with a disease or some other phenotype.

 Diagnostic use

 Assessment of associations between complex traits and both coding and noncoding variants
across the genome.

 Analyze, quantify and characterize circulating tumor DNA (ctDNA) in the bloodstream for early
cancer diagnosis, treatment selection and relapse monitoring, as well as for determining the
mechanisms of resistance, metastasis and phylogenetic patterns in the evolution of cancer.
• New born screening