Molecular diagnostics

Introduction to diagnostic lab setup, specimen

handling and safety
 Molecular diagnostic laboratory setup

– The correct workflow is followed in a molecular

laboratory in order to
• Minimize contamination and
• ensure good laboratory practices are followed.
– It is the responsibility of all laboratory staff to
ensure that the workflow is followed.
– PCR is extremely sensitive and thus poses a huge
risk of contamination.
– To minimize this and thereby reduce
contamination the
• Different areas in a molecular laboratory should be
physically separated
– Depending on the nature of the molecular assay
the ideal number of separations differs.
– Firstly, there should be two major separations
between the work done
• Prior to amplification (PRE-PCR)
– generally known as the clean area and
• After amplification (POSTPCR) generally known as the
dirty area
– Between these two areas the work flow should be uni-
directional
– the relative air pressure and direction should differ.
– The equipment, consumables and laboratory coats should
be dedicated to each area.
– If possible it is helpful to color code racks, pipettes and
laboratory coats in the different areas to be able to easily
monitor movement between the different areas.
– Furthermore, powder-free gloves should be used
throughout the process in all the different areas as the
power on powered gloves results in assay inhibition.
 Safety and specimen handling

– Molecular tests, like any clinical laboratory tests,

require
• optimal specimen handling and
• processing for accurate and consistent test results.
– The success of a test procedure is affected by the
• Age
• Type
• Condition of specimens.
– Therefore, specimen collection, transport, and
handling in the laboratory require careful attention.
– The clean area
• is divided into two additional areas, namely,
– Specimen processing laboratory:
» specimens are received, stored, total nucleic
acid is extracted and the generation of
complimentary DNA (cDNA) is performed.
– The non template laboratory
» reagents are stored and mastermix preparation
for cDNA and amplification are made
• Equipment's present in sample processing
laboratory
– - 80°C and -20°C freezers and a fridge for sample storage (depending
on the specimens received in the laboratory)
– a biohazard hood for sample extraction (especially if infectious
specimens are processed in the laboratory)
– a centrifuge (if required for specimen extraction)
– automated extraction platform
– a PCR workstation (a contained area that contains a UV light with or
without a timer)
– a thermocyler (for cDNA synthesis only)
– dedicated pipettes, dedicated vortex, a dedicated place to hang
laboratory coats and
– the appropriate safety materials (eye wash, medical aid box, shower).
– Equipment's present in non-template laboratory
• -20°C freezers and fridge for reagent storage
• dedicated pipettes, dedicated vortex, dedicated
microfuge
• a PCR workstation (a contained area that contains a UV
light with or without a timer)
• a dedicated place to hang laboratory coats and
• the appropriate safety materials (eye wash, medical aid
box, shower).
• Dirty area/room
– post-amplification
laboratory and the
nested PCR laboratory.
– The air pressure should
be slightly positive for
the nested PCR
laboratory and neutral
for the post-
amplification
laboratory and blow
into both the rooms.
– The post amplification laboratory is where
• The amplification reaction and
• Detection of amplicon occurs.
– The detection of amplification can occur on a
• Real-time PCR platform,
• Gel electrophoresis,
• ELISA based detection and
• Sequencing.
• Equipment present in dirty area
– -20°C freezer and fridge for amplicon and reagent storage
– a centrifuge (if required for the molecular assay performed)
– a PCR workstation (a contained area that contains a UV light
with or without a timer)
– any equipment required for amplification
– gel electrophoresis, sequencing or other amplicon detection
methodology
– dedicated pipettes, dedicated vortex, a dedicated place to hang
laboratory coats and
– the appropriate safety materials (eye wash, medical aid box,
shower).
• The lab layouts may depend on
– cost and space constraints
– It ensure there is as little movement between
clean and dirty areas
 General Step in Molecular
Laboratory

Gel
DNA extraction PCR / RT PCR Sequencing
electrophoresis
 IDEAL DESIGN MOLECULAR
LABORATORY
• 4 ROOM (Mandatory) • 3 ROOM • 2 ROOM
Reagen Preparation
Reagen
Preparation
Reagen Preparation
Nucleic Acid Extraction & Nucleic Acid
Nucleic Acid Extraction
Extraction
Amplification Amplification &
post amplification
Amplification &
Post
post amplification
Amplification/Sequensing Closed system
 Minimum Instrument in Molecular
Laboratory
• Reagen Preparation

Spin down Centrifuge

PCR Cabinet for mastermix
preparation
Minimum Instrument in Molecular Laboratory: Nucleic
Acid extraction Room
Biosafety Cabinet

Biohazard bin: layered with biohazard

bag,

Refrigerator & Freezer

Centrifuge and heat block

Micropippette & tips

Dedicated lab coats

Class I
Biosafety Cabinet for Molecular
Diagnostics

Class III
Class II
Minimum Instrument in Molecular
Laboratory: PCR & Post PCR room
Instruments & equipment PCR
 Example position Instrument of a Four-Lab
Layout
Ice
-20°C

Centrifuge
-20°C machine
sink freezer
freezer
BSC BSC

freezer
-80°C
workbench

LAB 1 LAB 2
Pass-
"No Template" through
"Specimen
workbench

Lab window Processing" Lab

freezer
-80°C
NA extraction
Lab coat rack Ice sink Lab coat rack Eye
equipment
machine wash

Lab coat rack Lab coat rack Eye

-20°C Ice
wash sink workbench machine
freezer
workbench

LAB 3 LAB 4
"Post-

freezer
-20°C
"Nested PCR"
Lab/Amplification amplification" Lab
Lab
Pass box
Centrifug

sink workbench PCR PCR

Sequencer Gel electrophoresis area
machines machines
e

WHO | HIV drug resistance laboratory training package. WHO, (available at

BSC: bio-safety cabinet https://www.who.int/hiv/pub/drugresistance/lab_training/en/).
Potential Sources of Contamination How to Controle
Cross
Amplification
contamination
product Laboratory design
between
contamination
specimens

Laboratory surfaces Ventilation ducts

Laboratory
practices

Hair, skin, saliva, Chemical and

Reagents/supplies and clothes of lab enzymatic controls
personnel

R. Lee, Molecular Lab design, 2015

 Basic Principe of Setting Up a Molecular
Laboratory
• Mechanical barriers to prevent contamination
• Spatial separation of pre- and post-
amplification work areas
• Area 1 – Reagent preparation
• Area 2 – Specimen/control preparation, PCR set-up
• Area 3 – Amplification
• Area 4 – Product Amplification detection
• Physically separated and, preferably, at a substantial distance
from each other
sticky mats
Unidirectional workflow

Reagent Specimen Amplification Sequencing

Preparation Preparation

0 105 1010 1012

Copy #

WHO | HIV drug resistance laboratory training package. WHO, (available at

https://www.who.int/hiv/pub/drugresistance/lab_training/en/).
 Unidirectional
Flow
Both personnel,
including Amplification product- Remove PPE before
cleaning personnel, & free to leaving
product-rich one area
specimens

Reusable supplies in
Avoid or limit reverse the
reverse direction need
direction to be
bleached

R. Lee, Molecular Lab design, 2015

Ideal Lab Workflow

SPECIMENS LAB 2
Frozen plasma "Specimen Processing" Lab
Whole blood Specimen receipt & storage
DBS RNA extraction and 1st round
PCR set-up

LAB 1 LAB 4
"No Template" Lab "Post-amplification" Lab
2nd round amplification
Reagent storage and
Gel electrophoresis
Master mix preparation
Sequencing reactions
Capillary electrophoresis
LAB 3
“1st Round PCR” Lab
1st round amplification
2nd round PCR set-up

WHO | HIV drug resistance laboratory training package. WHO, (available at

https://www.who.int/hiv/pub/drugresistance/lab_training/en/).
UV LAMP and Electronic Timer
Switches
Electronic Timer Switches

UV lamp
Three-Lab Setup PCR
Laboratory

3 2 1

http://www.lifesci.com/xcart/product.php?productid=16&cat=1&page=1
Three-Lab Setup (Option
1)
SPECIMENS LAB 1
Frozen plasma "Specimen Processing" Lab
Whole blood Specimen receipt & storage
DBS Master mix preparation

RNA extraction and 1st round

PCR set-up

LAB 3
"Post-amplification" Lab
Gel electrophoresis
Sequencing reactions
LAB 2 Capillary electrophoresis
"Amplification" Lab
1st round amplification
2nd round PCR set-up

2nd round amplification

In dead-air cabinet with dedicated pipettes

WHO | HIV drug resistance laboratory training package. WHO, (available at

https://www.who.int/hiv/pub/drugresistance/lab_training/en/).
Three-Lab Setup (Option
2)
LAB 2
SPECIMENS
Frozen plasma
"Specimen Processing and
Whole blood PCR" Lab
DBS Specimen receipt & storage
RNA extraction and 1st round
PCR set-up
1st round amplification
2nd round PCR set-up

LAB 1
"No Template" Lab
Reagent storage and
LAB 3
Master mix preparation "Post-amplification" Lab
2nd round amplification
Gel electrophoresis
Sequencing reactions
Capillary electrophoresis
In dead-air cabinet with dedicated pipettes

WHO | HIV drug resistance laboratory training package. WHO, (available at

https://www.who.int/hiv/pub/drugresistance/lab_training/en/).
Two-Lab Setup
LAB 1
SPECIMENS "Specimen Processing" Lab
Frozen plasma Specimen receipt & storage
Whole blood
Master mix preparation
DBS
RNA extraction and 1st round
PCR set-up

LAB 2
"Post-Amplification" Lab
1st round amplification
2nd round PCR set-up

2nd round amplification

Gel electrophoresis
Sequencing reactions
Capillary electrophoresis
WHO | HIV drug resistance laboratory training package. WHO, (available at
In dead-air cabinet with dedicated pipettes https://www.who.int/hiv/pub/drugresistance/lab_training/en/).
 Specimen handling
– Each laboratory will have requirements for
specimen handling, but general policies apply to
all specimen collection.
• A requisition or electronic test order should
accompany the specimen.
• The condition of the specimen and, if necessary, the
chain of custody is reviewed on receipt in the
laboratory
– No specimen is accepted
• without proper labeling and
• identification on the specimen tube or container
(placed by the person who collected the specimen),
• nor is a specimen accepted if the labeling on the
specimen does not match that on the accompanying
requisition.
– In addition to relevant patient identification, the
test requisition includes the
• type of specimen material (e.g., blood or bone marrow)
• ordered test
• date and time of collection
• reason for testing
• the contact information (pager or telephone number)
of the ordering physician.
– When required (for molecular genetics, forensics, or
parentage testing),
• Patient consent forms
• Ethnicity
• Photo identification of the individuals tested,
• Patient label verification
• Transfusion history or a pedigree may also be supplied with
the test specimen.
– Forensic specimens may require a
• Documented chain of custody.
• Bar coding of this information expedites specimen
accession.
– The laboratory should have written
• Procedures for documentation of specimen handling and
accession.
– Accession books or electronic records are used to
record the
• Date of receipt,
• Laboratory identifier, and
• Pertinent patient information associated with the
accession.
– If a specimen is unacceptable,
• The disposal or retention of the specimen is recorded in the
patient report or laboratory quality assurance records.
• If the specimen cannot be tested, the ordering physician is
notified.
– Molecular tests, like any clinical laboratory tests,
require optimal specimen handling and processing for
accurate and consistent test results.
– The success of a test procedure is affected by the
» Age
» Type
» Condition of specimens.
– Therefore, specimen collection, transport, and
handling in the laboratory require careful attention.
– If not processed immediately,
• Specimens are maintained in secure areas with limited
access under the appropriate conditions for the analyte
being tested.
Sample collection tubes for molecular testing

– Phlebotomy collection tubes are available with a number of

different additives designed for various types of clinical
tests.
– A selection of collection tubes commonly used for
molecular biology studies is listed in table below.
– Some anticoagulants used in blood and bone marrow
collection may adversely affect analytical results.
 Safety in diagnostic labs

• Precautions
– Standard precautions are recommended by the
centers for disease control and prevention for
handling potentially infectious specimens.
– All specimens are potentially infectious, so they
should all be handled with standard precautions using
proper personal protective equipment (PPE) to
prevent disease transmission.
– Transmission- based precautions including
respirators are used with airborne or contact-
transmissible agents.
– Contact precautions are designed for direct patient
care where there is the potential for direct exposure to
infectious agents on or from the patient.
– In general, standard precautions, including
• Gloves
• Gowns as PPE, are used by the molecular laboratory
technologist who has no direct contact with patients.
• Eye protection or masks are required in cases where
frozen tissue is being processed or where spraying or
splashing of a sample may occur.
– Gloves are important, not only as part of
• Standard precautions but also to protect nucleic acids
from nuclease degradation
CONTAMINATION IN MOLECULAR LABORATORY
• Introduction of unwanted nucleic acids into specimen
• the sensitivity of PCR techniques makes them vulnerable to
contamination
• Repeated amplification of the same target sequence leads
to accumulation of amplification products in the laboratory
environment
• A typical PCR generates as many as 109 copies of target sequence
• Aerosols from pipettes will contain as many as 106 amplification
products
• Buildup of aerosolized amplification products will contaminate
laboratory reagents, equipment, and ventilation systems
 Holding and storage requirements

– Methods such as interphase and metaphase

• Fluorescent in situ hybridization (FISH) and
• Karyotyping require intact cellular structures or
culture of cells so that only fresh specimens are
acceptable
– DNA and RNA are stable when samples are collected and
held under the proper conditions.
• For example, multiple tests may be performed on snap-
frozen tissue specimens held at –70°C.
 Molecular Diagnosis using the following
diagnostic
• PCR-RFLP (restriction fragment length polymorphism)
• SSP-PCR (single specific primer- PCR)
• MSP-PCR (Methylation-specific PCR)
• RT-PCR (Reverse transcription polymerase chain reaction)
• SSOP-PCR (sequence-specific oligonucleotides probes- PCR)
• STR (Short Tandem Repeats)
• VNTR (Variable Number Tandem Repeat)
• RFLP (restriction fragment length polymorphism)
• Immunohistocompatibility (IHC)
• Chromogenic insitu hybridization(CISH)
• ELISA
• Flow cytometry
• proximity assay
• telomerase activity assay
• Fluorescent in-situ hybridization (FISH)
• SKY (spectral karyotyping)
• M-FISH (Multicolor fluorescence in situ hybridization)
• CGH (comparative genomic hybridization)
• SNP marker analysis (single nucleotide polymorphisms marker analysis)
• DNA sequencing
PCR-RFLP (restriction fragment length
polymorphism)
 SSP-PCR (single specific primer- PCR)

• 1. the chromosomal DNA is digested with one or two

restriction enzymes
• 2 the unknown end of the restricted chromosomal DNA is
ligated to a suitable oligomer (Generic Oligomer) of known
sequence, sufficiently long to serve as a PCR primer
(Generic Primer); or the unknown end can be ligated to a
vector, in
• (3) the ligation reaction mixture is subjected to PCR
amplification using a specific primer annealing to the
known sequence and a generic oligomer annealing to the
unknown end
• permits amplification of genes for which only a partial
sequence information is available,
 MSP-PCR (Methylation-specific PCR)

• is used to detect genes or sequences with DNA methylation.

• During DNA replication, a methyl group is added on the pyrimidine
ring of cytosine on the 5 position (5-methylcytosine) in methylation
(CpG cytosines). This process is mediated by DNA
methyltransferases
• rapidly assess the methylation status of virtually any group of CpG
sites within a CpG island
• This assay entails the initial modification of DNA by sodium
bisulfite, converting all unmethylated cytosines to uracils but
leaving the methylated cytosines unchanged, followed by
subsequent amplification with primers specific for methylated vs
unmethylated DNA
• Methylation is one of the cause of cancer development in cells
RT-PCR (Reverse transcription polymerase chain
reaction)

• RNA is extracted, an aliquot of the extracted sample is added to a

reaction mixture which contains reverse transcriptase enzyme,
primers specific for the target of interest, and nucleotides.
• If the target is present, primers anneal to the RNA strand.
• Reverse transcriptase enzyme synthesizes a complementary DNA
strand, extending from the primer.
• The temperature is raised to 95o C, and the RNA/DNA strands are
denatured.
• The temperatures are lowered, allowing primers to anneal to the
newly formed cDNA.
• Polymerase enzyme synthesizes a new DNA strand, extending from
the primer.
• Multiple cycles geometrically increase the number of copies of
DNA.
RT- PCR
 SSOP-PCR (sequence-specific oligonucleotides
probes- PCR)

❖ DNA is extracted and quantified

❖ DNA is mixed with the cocktail of
primers, buffer, and Taq polymerase
for PCR/thermocycling.
❖ After PCR, the amplicons are
hybridized with probes/beads, added
to the amplicons, and subject to
washes, where unbound DNA and
debris are washed away.
❖ The labeled probes/beads are bound
with a fluorescent label for detection
and analysis.
❖ The product is then read by
instrumentation and interpreted using
software.
 STR (Short Tandem Repeats) analysis

• is a common molecular
biology method used to
compare allele repeats at
specific loci in DNA
between two or more
samples.
• A short tandem repeat is a
microsatellite with repeat
units that are 2 to 7 base
pairs in length, with the
number of repeats varying
among individuals, making
STRs effective for human
identification purposes
 VNTR (Variable
Number Tandem
Repeat) analysis
• VNTR or the Variable
Number of Tandem
Repeats are the repeated
DNA sequences at a
defined locus.
• The repeats are clustered
together and oriented in
the same direction.
• These can be found on
many chromosomes, and
they often show variations
in length.
• Each variant acts as an
inherited allele that allows
its use for identification.
 RFLP (restriction fragment length
polymorphism)

• is differences (or variations) among people in their DNA

sequences at sites recognized by restriction enzymes.
• Such variation results in different sized (or length) DNA
fragments produced by digesting the DNA with a restriction
enzyme.
 Immunohistocompatibility (IHC)

• Are group of genes that code for proteins found

on the surfaces of cells that help the immune
system recognize foreign substances.
• There are two major types of MHC protein
molecules—class I and class II. Class I MHC
molecules span the membrane of almost every
cell in an organism, while class II molecules are
restricted to cells of the immune system called
macrophages and lymphocytes
• Immunohistochemistry (IHC) uses antibodies to
detect antigens in a tissue sample
Chromogenic insitu hybridization(CISH)
• is a cytogenetic technique developed in the 1980s that
uses fluorescent probes to interrogate the genome of a
cell for the presence, quantity, and distribution of DNA
with a high degree of sequence complementarity.
• In situ hybridization (ISH) allows evaluation of genetic
abnormalities, such as changes in chromosome
number, chromosome translocations, or gene
amplifications, by hybridization of tagged DNA (or RNA)
probes with complementary DNA (or RNA) sequences
in interphase nuclei of target tissue
• CISH, or chromogenic in situ hybridization, is a process in
which a labeled complementary DNA or RNA strand is used
to localize a specific DNA or RNA sequence in a tissue
specimen.
• CISH methodology may be used to evaluate gene
amplification, gene deletion, chromosome translocation,
and chromosome number.
• CISH utilizes conventional peroxidase or alkaline
phosphatase reactions visualized under a standard bright-
field microscope, and is applicable to formalin-fixed,
paraffin-embedded (FFPE) tissues, blood or bone marrow
smears, metaphase chromosome spreads, and fixed cells.
 ELISA
• ELISA is the basic assay technique, known as enzyme-linked
immunosorbent assay (also referred to as EIA: Enzyme
Immunoassay) that is carried out to detect and measure
antibodies, hormones, peptides and proteins in the blood.
• Antibodies are blood proteins produced in response to a
specific antigen. It helps to examine the presence of
antibodies in the body, in case of certain infectious
diseases.
• ELISA is a distinguished analysis compared to other
antibody-assays as it yields quantitative results and
separation of non-specific and specific interactions that
take place through serial binding to solid surfaces, which is
normally a polystyrene multiwell plate.
ELISA
• Types Of ELISA
• ELISA tests can be classified into three types depending upon
the different methods used for binding between antigen and
antibodies, namely:
• Indirect ELISA - Indirect ELISA detects the presence of an
antibody in a sample.; The antigen is attached to the wells of
the microtitre plate.
• Sandwich ELISA - Sandwich ELISA helps to detect the
presence of antigen in a sample.; The microtitre well is coated
by the antibody.
• Competitive ELISA - Competitive ELISA helps to detect antigen
concentration in a sample. – Microtiter well which is antigen-
coated is filled with the antigen-antibody mixture
• Principle of ELISA
• ELISA works on the
principle that specific
antibodies bind the
target antigen and
detect the presence
and quantity of
antigens binding.
• In order to increase
the sensitivity and
precision of the assay,
the plate must be
coated with antibodies
with high affinity.
• ELISA can provide a
useful measurement
of antigen-antibody
concentration.
 Flow cytometry

• Flow cytometry is a lab test used to analyze characteristics of

cells or particles. During the process, a sample of cells or
particles is suspended in fluid and injected into a flow
cytometer machine.
• Approximately 10,000 cells can be analyzed and processed by
a computer in less than one minute.
• The flow cell (also called the flow chamber) allows the cells
(particles) within the samples to line up single file.
• This is a critical step for single-cell analysis.
• Flow cytometry may be used to characterize and count types
of white blood cells in the evaluation of infectious diseases,
autoimmune disorders or immunodeficiencies. It's also used
to diagnose and classify leukemia or lymphoma.
 Proximity assay
• It permits detection of
protein-protein interactions
in situ (at distances < 40
nm) at endogenous protein
levels.
• It exploits specific
antibodies identifying
(either directly or
indirectly) the two proteins
of interest and takes
advantage of specific DNA
primers covalently linked to
the antibodies
• A hybridization step
followed by a PCR
amplification with
fluorescent probes then
permit visualization of
spots of proximity by
fluorescence microscopy
Fluorescent in-situ hybridization (FISH)

• provides researchers
with a way to visualize
and map the genetic
material in an
individual's cells,
including specific genes
or portions of genes.
• This may be used for
understanding a variety
of chromosomal
abnormalities and
other genetic
mutations
 SKY (spectral karyotyping)
• Is advanced molecular cytogenetic
techniques for chromosome analysis
that are based on the principle of
FISH (fluorescence in situ
hybridization)
• is a karyotype in which the
homologous pairs of chromosomes
are manipulated in such a way that
they have distinctive colors.
• Spectral karyotyping and multicolor
FISH are techniques allow
visualization of all the chromosomes
simultaneously by labeling them
with a combination of different
colors that are spectrally
distinguishable fluorochromes, but
different methods are used for
detecting and discriminating the
different combinations of
fluorescence after in situ
hybridization
• the images are captured by charge-coupled device
(CCD) imaging and analyzed by using an interferometer
attached to a epifluorescence microscope.
• Image processing software then assigns a pseudo color
to each spectrally different combination, allowing the
visualization of the individually colored chromosomes
• analysis of abnormal karyotypes, unresolved by
conventional cytogenetics and the ability to identify
cryptic translocations in apparently ‘normal’
karyotypes
 M-FISH (Multicolor fluorescence in situ
hybridization)

• provides researchers with a way to visualize and map

the genetic material in an individual's cells, including
specific genes or portions of genes.
• This may be used for understanding a variety of
chromosomal abnormalities and other genetic
mutations.
• In M-FISH each homologous pair of chromosomes is
uniquely labeled with five fluorochromes set which are
spectrally distinct in different combinations.
• The images are captured by band-pass filter sets and
defined emission spectra are measured by dedicated
M-FISH software.
 CGH (comparative genomic hybridization)

• is a technique that permits the detection of

chromosomal copy number changes without the
need for cell culturing.
• It provides a global overview of chromosomal
gains and losses throughout the whole genome
of a tumour
• is based on the simultaneous hybridization of
differentially labeled test and normal reference
DNAs to normal metaphase chromosomes.
• The hybridized DNAs are detected with two
different fluorescent dyes
SNP marker analysis (single nucleotide polymorphisms
marker analysis)

• SNPs are found in the DNA between genes.

• They can act as biological markers, helping scientists locate
genes that are associated
• SNP markers are also the best choice for construction of a
dense set of polymorphic markers that can be used for
studying the association between the markers and a particular
trait or disease.
• at a genomic location with the sequence ACCTGA in most
individuals, some persons may contain ACGTGA instead. The
third position in this example would be considered an SNP,
since there is a possibility of either a C or a G allele occurring
in the variable position
• principle lies in hybridizing the sample DNA
with SNP probes on the chip and determining
genotypes at each SNP locus through the
signal strength of the hybridization between
the probe and sample DNA.
• SNPs have many potential uses in forensic
genetic investigations, including estimation of
ethnicity, human traits, or diseases
 DNA sequencing
• DNA sequencing refers to the general laboratory technique
for determining the exact sequence of nucleotides, or
bases, in a DNA molecule.
• The sequence of the bases (often referred to by the first
letters of their chemical names: A, T, C, and G) encodes the
biological information that cells use to develop and
operate.
• may be used to determine the sequence of individual
genes, larger genetic regions (i.e. clusters of genes or
operons), full chromosomes, or entire genomes of any
organism.
• DNA sequencing is also the most efficient way to indirectly
sequence RNA or proteins (via their open reading frames).
• DNA sequencing is used for a range of
purposes, including diagnosis and treatment
of diseases.
• In general, sequencing allows health care
practitioners to determine if a gene or the
region that regulates a gene contains changes,
called variants or mutations, that are linked to
a disorder
• See video
 Cycle sequencing

• is a simple method in which successive

rounds of denaturation, annealing, and
extension in a thermal cycler result in linear
amplification of extension products.