2. MAHRASHI DAYANAND UNIVERSITY
CELLULAR AND MOLECULAR BIOLOGY
[ PCR, DNA ELECTROPHORESIS, GENE SEQUENCING
DEPARTMENT OF PHARMACEUTICALSCIENCE
UNDER THE GUIDANCE
DR. ANURADHA PANNU
ASSISTANT PROFESSOR
MDU.
SUBMITTED BY:
TARANJUM
1803
M.PHARMACY
IST YR
4. DNA ELECTROPHORESIS
Electrophoresis is the migration of charged particles or molecules in a medium under the influence of an electric field.
DNA-DNAGel Electrophoresis (Agarose gel electrophoresis).
PROTEINS- SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis).
Ions that are suspended between two electrodes tends to trave towards the electrodes that bears opposite charges.Depending on kind
of charge the molecule carry, they move towards either .
▶
▶
▶
▶
▶
▶
▶
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To cathode Or to Anode
AN ELECTROPHORESIS
A separation technique, determine the size of DNA Fragments.
Simple, rapid and highly sensitive.
Used in clinical laboratories to separate charged molecules from
5. Principle
▶ Comprehensive term that refers to the migration of charged particles of any size in liquid media under the influence of
an electric field.
▶ Depending on kind of charge the molecule carry, they move towards either
▶ • To Cathode or to Anode
▶ An ampholyte become positively charged in acidic condition and migrate to Cathode, in alkaline condition they become
negatively charge and migrate to Anode.Shorter molecules move or migrate faster than longer ones.
▶ The rate of migration of an ion in electrical field depend on factors,
▶ 1.Net charge of molecule
▶ 2.Size and shape of particle
▶ 3.Strength of electrical field
▶ 4. Properties of supporting medium
▶ 5. Temperature of operation
6. How Separation Occurs
Electric charge
▶ Many molecules (amino acids, proteins, DNA,
and RNA) have naturally occurring negative
and positive charges on them.
Molecules with a positive charge (cations) will
be attracted to the negatively charged node
(cathode).
Molecule size
▶ The porous material is made of microscopic
particles suspended in a gel.
▶ The microscopic particles attach to one
another forming tunnels that act as a sieve to
separate the molecules.Small molecules can
move faster than large molecules.
7. Materials required for gel electrophoresis
▶ Electrophoresis chamber
▶ Agarose gel
▶ Gel casting tray
▶ Buffer Staining agent (dye)
▶ Comb
▶ DNA ladder
8. Procedure of Gel Electrophoresis
▶ Step 1:
▶ An Agarose and buffer solution is poured into a tray and chamber.
▶ A Comb is placed into the tray on one end
▶ Step 2:
▶ The Agarose polymerizes into a gel as it cools.
▶ The Comb is removed from the gel to form Wells for samples.
▶ Step 3:DNA Samples coloured with a tracking dye are pipette into the Wells.
9. Step 4:
The tray is placed in a chamber that generates electric current through the gel.
The negative electrodeis placed on the nearest side of the samples.
The positive electrode is placed on other side.
Step 5:
DNA has a negative change and move towards the positive electrode
Smaller DNA molecules will be able to move faster than longer moleculesthrough the gel.
Step 6:
One well, called a DNA ladderwill contain DNA fragments of known sizes.
This ladder is used to determine the sizes of other samples.
10. Types of Electrophoresis
▶ Agarose Gel
▶ A porous material derived from red seaweed Agarose
is highly purified to remove impurities and charge
Acts as a sieve for separating molecules. This solid
matrix will allow the separation of fragments by size.
▶ Concentration affects molecules migration
▶ Low conc= larger pores→ better resolution of larger
DNA fragments
▶ High conc. = smaller pores→ better resolution of
smaller DNA fragments
11. Fragment Resolution
Gel Concentration - Is dependant upon the size - of the DNA fragments to be
separated.
% Agarose DNA fragment
▶ 0.5 30- 1
▶ 0.7 12- 0.8
▶ 1.0 10- 0.5
▶ 1.2 7- 0.4
▶ 1.5 3-0.2
▶ Agarose at Room Temperature is a 3-Dimentional solid matrix.The smaller the fragments the further the migration or
movement through the matrix.
▶ Purposes for Agarose Gel Electrophoresis
▶ Analysis of molecules size
▶ Separation and extraction of molecules
▶ Quantification of molecules
12. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE):
It is prepared by polymerizing acryl amide monomers in the presence of methylene-bis- acrylamide to cross
link the monomers.
▶ Structureof acrylamide (CH=CH-CO-NH₁)
▶ Polyacrylamide gel structure held together by covalent cross-links.Polyacrylamide gels are tougher than
agarose gels.
▶ It is thermostable, transparent,strong and relatively chemically inert.
▶ Gels are uncharged and are prepared in a variety of pore sizes.Proteins are separated on the basis of charge to
mass ratio and molecular size, a phenomenon14 led Molecular sieving.
▶ Types of PAGE
▶ PAGE can be classified accordingthe separation conditionsinto:
▶ NATIVE-PAGE:Native gels are run in non-denaturing conditions, so that the analyte's natural structure is
maintained.Separation is based upon charge, size, and shape of macromolecules.Useful for separation or
purification of mixture of proteins.Thiswas the original mode of electrophoresis.
▶ DENATURED-PAGE OR SDS-PAGE:Separation is based upon the molecular weight of proteins.The
common method for determining MW of proteins.Veryuseful for checking purity of protein samples.
13. Latest Advancement:
Global ElectrophoresisTechnologyMarkets to 2023
The study encompasses major technologies of electrophoresis and the various product segments for each technology. The role of
electrophoresis as an analytical separation method for both nucleic acid and protein molecules was examined.
This research analyzes the impact of the two major electrophoresis techniques on biological molecular separation,determines
their current market status,
▶ The report includes:
▶ 37 data tables and 22 additional tables
▶ Industry analysis of the electrophoresis technology, as a standardized analytical separation method
▶ Analyses of global market trends, with data from 2017 to 2018, and projections of compound annual growth rates
(CAGRs) through 2023
▶ Discussion of current market dynamics and future market potential for both gel electrophoresis (GE) and capillary
electrophoresis (CE) technologies
▶ Coverage of electrophoresis techniques and technologies used in academia, government research laboratories, and the
biopharmaceutical, environmental, chemical, forensics, and food industries
▶ Evaluation of the impact of lab-on-a-chip electrophoresis on the overall capillary electrophoresis technology segment
14. Future use of gel electrophoresis
▶ Antibiotics testing
▶ Data Analysis
▶ Vaccine testing
▶ Analysis of
proteins and
Vitamins
15. PCR (POLYMERASE CHAIN REACTION)
▶ PCR is a fast in vitro Enzymatic process for amplification of DNA.
▶ It was Invented by Kary Mullis in 1964.
▶ PCR has become one of the most commonly employed techniquesin molecular biology.
▶ It is a molecular technology aim to amplify a single or few copies of the DNA to thousands
or millions of copie.
▶ PCR is now a common and often indispensable technique used in medical and biological
research labs for a variety of applications.
▶ These include diagnosis of infectious diseases, DNA sequencingand DNA-based phylogen
▶ 1993, Mullisular technology aim to amplify a single or few copies of the DNA to thousands
or millions of copies.
16. CHARACTERISTICS FEATURES OF PCR
Sr.No Characteristics
features
Comments
1. Sensitivity Requirement of Initial
DNA is very less.
2. Specificity Only Desired DNA is
amplified.
3. Speed Very fast Amplification;
takes less than an hours
to few hours to
complete PCR.
17. Why "Polymerase"?
Because the only enzyme used in the reaction is DNA polymerase.
Why "Chain" ?
Because the products of the first reaction become the substrates of the following one and so
on.
▶ PCR Requirementsfor 25 micro litres
▶ DNase free water- 16.4 uL
▶ Magnesium chloride: 50mM
▶ Buffer: pH 8.3-8.8 - 2uL
▶ dNTPs: 10mM - 0.4uL
▶ Primers: 1.5uL
▶ DNA Polymerase: 1-2.5 units
▶ Target DNA: ≤ 1 μg
▶ The "Reaction"Components
▶ 1) Target DNA- contains the sequence to be amplified.
▶ 2) Pair of Primers - oligonucleotides that define the sequence to be
amplified.
▶ 4) Thermostable DNA Polymerase enzyme that catalyzes the reaction
▶ 5) Mg++ ions - cofactor of the enzyme
▶ 6) Buffer solution – maintains pH and ionic strength of the reaction
solution suitable for the activity of the enzyme
18. Steps of PCR
Steps Temperature Features Time
Denaturation 90- 95°C Denaturation of DNA
(dsDNA –ssDNA)
1 minutes
Annealing 55-60°C Attachment of primers to
the template
1 minutes
Extension 72°C Addition of dNTPs to
extend the complimentary
strand.
1.5 minutes
21. Efficiency of PCR
▶ The Efficiency of a PCR dependson following factors:
▶ Initial amount of DNA.
▶ Numbers of cycles
▶ Final amt of DNA.
▶ The formula used for calculationsof its efficiency is
▶ N = n(1+E)c
▶ N= Final amt of DNA, n= initi amt of DNA; E = Efficiency(0.70-0.93);c= number of cycles.
▶ For example: for amplification of approximately10 ▪9 fold of the sample DNA, at least 30 cycles
followed by ab extended 10 min round for DNA Synthesis will be required.
24. Real time PCR
▶ Steps:
▶ Add DNA Sample
▶ Add desired Primer and Probe
▶ Probe is short sequence complementary to DNA Like Primer
▶ One Side of Probe is Fluorescent Molecule while on Other end
Quencher is Present.
▶ Run PCR
▶ Fluorescent Molecule emitting Fluorescent light with each copy
Completing
▶ Probe is between Two Primers
▶ And this Fluorescent intensity detected by the Fluorescent detector
in the PCR
▶ Graph developed to determine the copies at any point of PCR
25. Advantages and Disadvantages of PCR
Advantages Disadvantages
High sensitivity compared to culture and staining Potentially lower specificity compared to culture and staining
Ability to testfor anti-microbial resistance Need for narrow list of causative agents to use specific primers
Quickly performed in 4-8 hours Possibility of amplifying normal flora from corneal scrapings
Shownto be more cost-effective with selective use than culture and
staining
Becomes less cost-effective when performed with a multi-organism
PCR approach
Increasedability to detect less common organisms such as viruses Supply costs, machinery fees, training expenses
26. ▶ OUTSTANDING CHALLENGES IN PCR
▶ Despite the great diagnostic potential of PCR, the success of each of its practical applications is highly dependent on the
quality of samples containing nucleic acids for amplification.
▶ Additionally, the successful PCR amplification of GC-rich DNA sequences represent another challenge. It is complicated
by the generation of secondary structures hindering full denaturation and primer annealing and represents multiple
techniques to overcome this problem have been established.[67].
▶ PCR COMMERCIALIZATION: HISTORY & SUCCESS
▶ There are two aspects to the commercialization efforts that have made PCR a gold standard in molecular diagnostics.
One is the development of an automated instrument replacing manual operations and the other is the set of reagents that
increase the PCR’s specificity, eliminating cross-contamination and inhibition, reducing reaction time and increasing
multiplexing capabilityt
▶ TECHNOLOGIES THAT EVOLVED FROM & WERE INSPIRED BY PCR:
▶ Digital PCR
▶ Many technologies have evolved from original PCR; one example is dPCR, which is based on splitting a PCR sample
into thousands, in some cases millions, of subsamples from the original to digitize the pool of DNA molecules having
either a single or no copy in each subsample. dPCR is always based on microfluidics
27. Chip-based digital PCR (cdPCR)
The sample is loaded into silicon chips with wells made by a micromachining technique. Then the thermal cycling is performed and the
chip is imaged by fluorescence microscopy to determine the number of wells with positive PCR results.Multiplexing is also performed in
the same way as in ddPCR.
Isothermal amplification
qPCR is conducted by thermal cycling and its rate is typically limited by sample cooling, which slows with increased sample volumes.
RECENT ADVANCES AND CHALLENGES OF RT-PCR TESTS FOR THE DIAGNOSIS OF COVID-1
Changing RT-PCR towards a point-of-care test for COVID-19
Given the aforementioned difficulties of the RT-PCR test, enormous efforts have been made to produce an easier, faster,
and more convenient test capable of being used outside the laboratory environment.
New 'nanoPCR' technology can accurately diagnose COVID-19 in less than 20 minutes
A "nanoPCR" technology was developed for the point-of-care (POC) diagnosis of coronavirus disease-19 (COVID-19). This new
technology can diagnose the infection within ~20 minutes while retaining the accuracy of conventional reverse transcription polymerase
chain reaction (RT-PCR) technology.
A team of researchers led by Professor CHEON Jingo, the director of the Centre for Nano medicine (CNM) within the Institute for Basic
Science (IBS) in Seoul,
28. Application of PCR
Molecular
Identification
• DNA fingerprinting
• Classification of organisms
• GenotypingPre-natal diagnosis
• Mutation screening
• Drug discovery.
• Genetic matching
• Detection of pathogens
Sequencing Genetic
Engineering
• Gene expression studies.
• Site directed mutagenesis
• Bioinformatics
• Genomic cloning
• Human Genome Project
29. GENE SEQUENCING
▶ It is also known as DNA sequencing.
▶ Gene sequencing may be defind as it is a process of determining the nucleic acid sequence-the order of nucleotides in DNA.
▶ It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and
thymine.
▶ Need of sequencing
▶ Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development
of treatment.
▶ An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or
animal.
▶ Simple point mutations can cause altered protein shape and function.
30. Generation of Gene Sequencing
1st Generation sequencing:
▶ Maxam- Gilbert sequencing
▶ Sanger sequencing.
Advance Generation sequencing (shotgun)
▶ Whole genome shotgun
▶ Double barrel shotgun
▶ Hierarchical shotgun
Next Generation sequencing
▶ Sequencing by ligation
▶ Pyrosequencing
▶ Single molecular real time sequencing
31. The Maxam-Gilbert technique :
▶ Principle
▶ Chemical Degradation of Purines and pyrimidines
by dimethylsulphate and hydrazine respectively and
then labeled it
▶ Aliquot A+ dimethyl sulphate, which methylates
guanine residue
▶ 2.Aliquot B + formic acid, which modifies adenine
and guanine residues
▶ 3. Aliquot C + Hydrazine, which modifies thymine
+ cytosine residues
▶ 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which
makes the reaction specific for cytosineGCTAC
Labeled fragmentTA11.
32. Sanger Chain Method
▶ The key principle of the Sangers method was use of dideoxynucleotides triphosphate (ddNTPs) as DNA chain
terminator
▶ It involves following components:
▶ 1. Primer
▶ 2. DNA template
▶ 3.DNA polymerase
▶ 4.dNTPs(A,T,G,C)5. ddNTPs
▶ • It involves following 4 Steps:
▶ 1. Denaturation
▶ 2. Primer attachment and extension of bases
▶ 3.. Termination
▶ 4. Poly acrylamide gel electrophoresis
33.
34. The Methods (Procedure):-
1. Before the DNA can be sequenced,it has to be denatured into single strands using heat.
2. Next a primer is annealed to one of the template strands.
This Once the primer is attached to the DNA, the solution is divided into four tubes labeled "G", "A", "T" and
"C".
▶ "G" tubes: all four dNTP's, ddGTP and DNA polymerase
▶ "A" tubes: all four dNTP's, ddATP and DNA polymerase
▶ “T" tubes: all four dNTP's, ddTTP and DNA polymerase
▶ "C" tubes all four dNTP's, ddCTP and DNA polymerase
▶ Mixture first heated so that DNA strands separate (96°C)
▶ Then temperature get lower so that short length DNA sequencea primer can bind to the template
DNA(50°C)
▶ Temperature raised to (60°C- 65°C)to enable the DNA Polymerase enzyme to bind to the short
section of doublestranded DNA.DNA polymerase to synthesizes new DNA.
▶ DNA Polymerase will continue adding Nucleotidesto chain until it happensto add a dideoxy
nucleotide instead of normal one
35.
36. Advance Generation sequencing
Double barred shotgun
Double-barrelshotgun sequencingis also
referred to as "pairwise-end sequencing".
Same as Whole-genome shotgun with one
difference.
Sequencingis performed from both ends of DNA
inserts as oppose to just one. Method conceived
to reduce "Gaps" and to reduce assembly error.
DISADVANTAGE:More amount of data is
generated so, it is difficult to assemble
ADVANTAGE: Theoreticallyit is very
accurate.
Hierarchicalshotgun:Hierarchical Shotgun Sequencing
Method
DNA is obtainedand cut into pieces of 150Mb
Piece inserted into bacterial clones(BACs).
BACS are then grown and the inserts recovered
Insert are then mapped to determine physical location
Inserts are then organized by known location="Golden
Tiling Path".)
Inserts are then fragmented, recovered, and sequenced
according to order in the "Golden Tiling Path“
38. ▶ Next Generation Sequencing
▶ Traditional DNA sequencing methods lack the capacity to provide genome analysis at speeds that the biotech sector
demands. This is where next-generation sequencing aids researchers and biotech companies
▶ . Startups are advancing a range of NGS technologies to enable ultra-high throughput and scalable genome sequencing.
This is enabling rapid sequencing of whole genomes, deep sequencing of target regions, and faster human genome
analysis. Moreover, NGS solutions greatly accelerate drug discovery workflowsand enable precision medicine.
▶ Helaxy offers Fluidics NGS Prep
▶ Helaxy is a Singaporean startup that develops a fluidics NGS prep solution. It features flexible sample and assay setup as
well as minimizes hands-on time to mitigate manual pipetting errors. Additionally, the solution uses generic reagent
cartridges to reduce reagent wastage. This allows hospitals to leverage decentralized COVID testing and move testing
closer to the patients, saving time.
▶ CD BioSciences enables NGS Library Synthesis
▶ CD BioSciences is a US-based startup that creates htDNA-chip, a DNA synthesis platform for NGS. It assists researchers
in preparing NGS libraries efficiently and makes millions of nucleotide chains in a single run.
Next generation sequencing
39.
40. Single Molecules Real time sequencing
▶ Single molecule real time
sequencing utilizes the zero-mode
waveguide (ZMW)A single DNA
polymerase enzyme is affixed at
the bottom of a ZMW with a single
molecule of DNA as a template
▶ Each of the four DNA bases is
attached to one of four different
fluorescent dyes.SMRT chip
contains ~3000 ZMW holes
41. Nanodrop
▶ The NanoDrop ND-1000 is a full- spectrum
(220-750nm) spectrophotometer that measures
1 ul samples with high accuracy and
reproducibility.
▶ Nucleic acid concentration and purity of
nucleic acid samples up to 3700 ng/ul
(dsDNA) without dilutionFluorescent dye
labeling density of nucleic acid microarray
samples
▶ General UV-Vis spectrophotometry
▶ 1 ul only of sample requiredLarge dynamic
range: 2- 3700 ng/ul of dsDNA
▶ DNA, RNA, microarray labeling dyes, and
proteins
42. Progress in DNA sequencing till the past decade and into
the future
44. References
▶ Heather, J. M., and Chain, B. (2016). The sequence of sequencers:The history of sequencingDNA. Genomics 107, 1–8.
doi: 10.1016/j.ygeno.2015.11.003
▶ Javan, G. T., Finley, S. J., Abidin, Z., and Mulle, J. G. (2016). The Thanatomicrobiome:AMissing Piece of the Microbial
Puzzle of Death. Front. Microbiol. 7:225. doi: 10.3389/fmicb.2016.00225
▶ Green MR, Sambrook J. Polymerase chain reaction (PCR) amplification of GC-rich templates. Cold Spring Harb.
Protoc. 2019(2), pdb. prot095141 (2019).
▶ AhrbergCD, Manz A, Neuzil P. Palm-sized device for point-of-care ebola detection. Anal. Chem. 88(9), 4803–4807 (2016).
▶ Jain M, Olsen HE, Paten B, Akeson M. The Oxford Nanopore MinION: delivery of nanopore sequencingto the genomics
community. Genome Biol. 17(1), 239 (2016).
▶ Tian H, SunY, Liu C, Duan X, Tang W, Li Z. Precise quantitation of microRNAin a single cell with droplet digital PCR
based on ligation reaction. Anal. Chem. 88(23), 11384–11389 (2016).
▶ Katsanis S.H. and Katsanis N. (2013) Molecular genetic testing and the future of clinical genomics. Nat. Rev. Genet. 14,
415–426 10.1038/nrg3493
▶ Caulfield M., Davies J., Dennys M., Elbahy L., Fowler T., Hill S.. et al. (2015) The 100,000 genomes project protocol.
