 Initial method that is being performed.
Primers
 Method that generates enough copies of
single gene sequence; increases the yield of
nucleic acid sequence of interest for
subsequent analyses.
o Specific strands that will be multiplied
 Identify the sequence
o Specific genes for every material
/
1983 First used by Kary Mullis in 1983 in
California.
Former the Polymerase-catalyzed
chain reaction.
Polymerase has an activity of
synthesizing new DNA strand.
Escherichia coli First successful amplification was
plasmid pBR322 a short fragment of Escherichia
coli plasmid pBR322.
Sickle cell anemia First application for diagnosis is
patient with sickle cell anemia.
 Uses DNA polymerase to drive the
replication and to catalyze the addition of
nucleotides to the growing strand with a use
of primer that adds subsequent bases.
 To generate millions of copies of target
sequence.
 In vivo - constant body temperature
o Readily available nucleotides
o Involvement of primase enzyme
o Own polymerase enzyme
 PCR - in vitro version of DNA synthesis
o Several temperature changes
o Involvement of nucleotides in the form of
reagents
o Primers
o Polymerase
o It was mirrored from the in vivo DNA
replication
 Directs DNA synthesis to the desired regions;
critical component as it determines the
specificity of the entire sequence to amplify.
 ssDNA with 20-30bp in length and in 2 set (1)
forward primer (2) reverse primer.
 Melting temperature (Tm) determines the
starting point of the primer as it affects the
binding of the primer to the sequence (50-70°
C).
o Parallel to the primase in vivo
o Primers, primase and probes same action
o Initial reaction  recognize specific
sequence
o They are often known sequence
Ex. COVID 19
o Appropriate temperature for it to work
DNA Template
 dsDNA or ssDNA derived from patient’s sample
or microorganism causing infection.
 Routine analysis: 100ng-1ug of DNA is used as a
template for amplification.
 Templates with high GC content and secondary
structures may encounter difficulty in
amplification and may require modifications.
o Regardless of the type of DNA it should be
denature before proceeding to actual
procedure.
o GC- triple bond
Deoxyribonucleotide bases
 Nucleotide triphosphates are the building blocks
of DNA.
 dNTPs: deoxynucleotidetriphosphates
o Adenine – deoxyadenosinetriphosphate
o Guanine – deoxyguanosinetriphosphate
o Thymine – deoxythymidinetriphosphate
o Cytosine – deoxycytidinetriphosphate
 In vitro - reagents and included in
master mix.
 Base that are being added when
synthesis happens or the polymerase will
continue ng nasimulan ni primer.
  Normal events: always triphosphate
DNA Polymerase
 Thermostable enzymes
o Taq polymerase from Thermus aquaticus
o Tth polymerase from Thermus thermophilus
 Stoffel fragment
o Modified Taq polymerase that lacks N-
terminal 289 amino acid.
o Has broader range of magnesium chloride
concentration than Taq.
o Ideal for amplifying regions with high GC
content.
 Responsible for synthesizing
 Primase to polymerase
PCR Buffer
 Provides optimal condition for enzyme activity;
increasing salt concentration makes longer DNA
products denature more slowly than shorted
DNA products.
 Maintains the proper pH of the buffer
o Potassium chloride: 20 – 10mM
o Ammonium sulfate: 15 – 30mM
o Magnesium chloride: 1 – 4mM
o Tris-HCl: proper buffering pH 8 – 9.5
 Accessory buffer – can be use or not
o Bovine serum albumin
o Dithriothreitol: 0.01mM
o Formamide: 1 – 10%
o Chaotropic agents: triton X-100, glycerol,
dimethyl sulfoxide: 1 – 10%
PCR Controls
 Running controls is essential for maintaining and
ensuring accuracy of the assay.
 Positive control ensures that the enzyme is
active, buffer is optimal, and primers are priming
right sequences.
o It should be both known properties
o Matinong reaction
 Negative control is without DNA that ensures all
reaction mix is not contaminated and primers
are not annealing to unintended sequences.
o Primer only, no DNA
o No reaction
o Nagkaroon ng reaction indicates
contamination
 Internal control more concern with the sensitivity,
ensure that every reaction is specific, often
associated with positive control groups.
Thermal Cyclers
 Machine that will provide temperature needed to
facilitate the process.
 Designed to ramp through the required
incubation temperatures rapidly and
automatically in several cycles.
o Rapid PCR: small sample volume
o Real time PCR: equipped with fluorescent
detectors to measure the product
1. Denaturation
 dsDNA is denatured into two single strands
 Temperature: 90-96°C (Buckingham), 94°C (S)
 Time: 20-60 seconds
o Mirrors the activity of the helicase.
2. Annealing
 Most critical step for the specificity of the PCR.
 Attachment of primers to the strand to be
amplified.
 Temperature: 50-70°C (B) 50-60°C (S)
 Time: 20-90 seconds
3. Extension
 DNA polymerase catalyzes the formation of
phosphodiester bond to replicate DNA by
extending the primers.
 For every cycle of a one dsDNA, it will be
replicated into two dsDNA.
 Temperature: 68-75°C (B) 72-74°C (S)
 Time: 10-60 seconds
 cDNA; enzyme is inactivated at 80°C once
reaction is fulfilled.
 Uses: gene expression analysis

Real-time/Quantitative PCR Method/qPCR

 Requires dsDNA-specific dyes such as ethidium

bromide or SYBR green that is added to the
sample before the PCR test; provides a
fluorescent signal as evidence of amplification.
o Exponential phase: fluorescence occurs
during reaction
o Threshold cycle (CT)
 Number of cycles required for
fluorescent signal to cross the threshold.
 Optimal level: based on the background
or baseline fluorescence and the peak
 1 cycle  1dsDNA = 2 dsDNA
fluorescence in the reaction.
 30 cycles- recommended for PCR
o Threshold value is inversely proportional to
 Conventional PCR (1set= 1 forward, 1 primer), 1
DNA concentration.
specific primer
 Low CT value = high DNA concentration
 High CT value = low DNA concentration
 Uses: gold standard for SARS-Cov-2 in
Multiplex PCR Method combination with RT-PCR.
 RT-qPCR/ qRT-PCR - gold standard for SARS-
 More than one primer pair can be added to a
Cov-2 or COVID-19
PCR for multiple amplifications are primed
simultaneously resulting in the formation of
multiple products.
 Uses: Identification analyses
o Adding of 3 known primer on patients
sample
o Different and many primers

Nested PCR Method

 Increased sensitivity by using two pairs of

primers to amplify a single target in separate
run.
 Uses: specimen with limited quantity and quality
o 1st pair= 1st round
o 2nd pair= proceeding rounds

Reverse Transcriptase PCR Method/RT - PCR

 Amplification method is modified to include the

initial incubation at 45-60°C for 30-60 minutes
which makes reverse transcriptase able to
convert any RNA sample to cDNA template.
 Reverse transcriptase: enzyme that converts any
RNA sample to become dsDNA in the form of
 strand  polymerase attach in SDA
 Another way to amplify the segments of primer  synthesizing new
nucleic acids. sequence  bumper primer
 It can be ssDNA or RNA (babangain) and will synthesize own
 Generates signal strand  SDA strand will be
 Synthetic probes bind to specific target displaced
sequences and probes are amplified.  displaced SDA  new polymerase to
 Methods: Ligase Chain reaction, Strand form dsDNA again  restriction site
Displacement amplification, Q-beta (restriction enzyme)
replicase  Displace strand has restriction site
o Exponential target amplification
 Goal: generate newer target strand
 Generates a signal by repeated ligation of probes  Restriction enzymes create a nick on the
complementary to specific sequence. target sequence, generating a substrate
 Instead of DNA polymerase synthesizing new for the polymerase and extend it; the
strand by the help of primers, DNA ligase is used displaced strand is hybridized by a
to ligate the adjacent primers bound to template primer producing another target
forming a new DNA strand. sequence with hemi sensitive restriction
o Kailangan strands na agad then check if its site.
complementary then it will bind  Restriction enzyme will cut the
o Use for restriction and plasmids restriction site forming nicks (gaps)
 forming new displacement 
Strand Displacement Amplification
polymerase forming new strand 
 Isothermal amplification method exonuclease, copy reading
o No need for thermocycler
o Single temperature requirement
 Uses two pairs of primers and can recognize two
regions of target sequences
o Bumper primer: similar to PCR primers
o SDA primer: bound at target sequence
 Possess a restriction site
 Can be in the form of probe
 Displaced strand
 Two steps method
o Target generation phase
 Goal: generate new target sequence
 Possess restriction site
 Involves denaturation of the DNA
template at 95°C; after denaturation
SDA primer and Bumper primer binds to
 Signals are bound to the target sequences
specific sequence of denatured DNA,
present in the sample; ideally performed for
new strand is formed from the two
quantitating amount of target sequences
primers thru DNA polymerase BUT the
instead of producing multiple copies.
strand produced by SDA primer will be
Branched DNA Amplification
displaced from the template.
 dsDNA  denature (separate)   Requires (1) capture probes (2) extender probes
attachment of two primer in one (3) pre-amplifiers
  Target sample is captured or immobilized to a
solid support by capture probes, after which
extender probes and blocking probes create a
stable cruciform structure.
o Media  capture probe must be specific
with the RNA that needed to be isolate 
sandwiching of extender probes 
pre amplifiers to catch all the amplifiers that
is responsible for the signals
o Signal indicate that there is bound nucleic
acid
o Higher signal means there is nucleic acid
o Branching from one probe to another
o Blocking probes to prevent unintentional
binding
o Capture probe catches DNA/RNA 
extender probe will attach to the
complementary DNA/RNA  amplifiers will
catch all the signal products to generate
signal that there is nucleic acid
o For detection, goal is to detect the number
 Uses: quantitative detection of HBV, HCV, and
HIV-1

Hybrid Capture Assay

 Starts with hybridization of RNA probe to the

denatured DNA target with hybrid-specific
immobilized antibodies; the binding of secondary
antibodies to RNA probes generates signal in the
presence of chemiluminescent substrate
o antibodies will capture but probe will attract
RNA  hybridize RNA probes with DNA 
going to antibody capture  incorporate
RNA probes that will generate a signal
(attachment of secondary antibodies) 
secondary antibodies has the signal (ALP) 
11 showing chemilluminescent signal
indicating presence of nucleic acid
o Antibodies are specific with RNA probes,
detect DNA
o Signal means it has nucleic acid
o Direct relationship
o Higher signal induction means many nucleic
acid has bind
 Uses: detection of HPV and CMV
  DNA sequencing is performed to determine

4 minutes

5 minutes

8 minutes

8 minutes
the order of nucleotides in a DNA molecule
 The information is used for detecting

°
mutations, designating polymorphism,
identifying haplotypes, typing

Splits pyrimidine
Protonates purine
microorganism etc.

Splits cytosine
Methylates
guanine

rings

ring
 Most specific for identifying genetic mutations in

Hydrazine with salt

DNA polymer

Dimethylsulphate

Formic acid
 Two method: Maxam-Gilbert Sequencing and

Hydrazine
Sanger Sequencing

Maxam-Gilbert Sequencing

 Developed by Allam Maxam and Walter Gilbert

in the year 1970s

Guanine and

Thymine and
Cytosine

Cytosine
Guanine

Adenine
 Based on the migration of the bands in relation to
its size with 5’ end consider as the smallest
region and 3’ having the largest region; implies
the principle of gel electrophoresis
o 5’ near the bottom and 3’ near the specimen Procedure:
well
1. dsDNA is denatured into ssDNA
 Components
2. Prepare four test tubes and pipette your ssDNA
o dsDNA or ssDNA template
sample to each test tube
o 10% piperidine as reducing agent
3. Add the required chemical on each test tube
o Base modifies (1) dimethylsulphate (2) formic
a. Test 1 add Dimethylsuphate
acid (3) hydrazine (4) hydrazine with salt
b. Test 2 add Formic acid
o 6% polyacrylamide gel for separation of
c. Test 3 add Hydrazine salt
fragmented DNA
d. Test 4 add Hydrazine
o Loading dye for gel electrophoresis
4. Add the reducing agent at 90C to facilitate
 DNA template is aliquoted into four tubes each
breaking of the strand
with specific base modified added together with
5. Proceed to polyacrylamide gel electrophoresis to
the reducing agent to effectively facilitate
separate the fragments based on their size
cleaving at specific nucleotide level; afterwards,
6. Proceed to autoradiograph to transfer the bands
the sample will be treated and proceed with gel
to a X-ray film to be observed
electrophoresis for separation
o Tubes that detect single base nucleotide will
 The entire process lasts for 1-2 hours for short
serve as a control to tube that has 2
fragments and 7-8 hours for longer fragments
nucleotides
o Appearance of bands
 o Tube for each ddNTP

Approaches to Sequencing

 Based on the principle of Sanger sequencing

 Label fragments synthesized according to
 terminal end of the sample
Nucleotide  1 tube with color designation
1st T  Polymerase needed to work, DNA
2nd G synthesis first before ddNTP can be
3rd C added
th
4 A o ddATP – read as A in the termination
th sequence
5 T
6 th
T o ddCTP – read as C in the termination
7 th
C sequence
8 th
A o ddGTP – read as G in the termination
th sequence
9 A
o ddTTP – read as T in the termination
10th G
sequence
Sanger Sequencing
 Methods of Sequencing
 Discovered by Frederick Sanger in the year 1977 o Dye primer
 Based on the addition of a nucleotide that lacks a  Four different fluorescent dyes are
3’ hydroxyl ends that terminates the addition of attached to four separate aliquots of the
new nucleotides to stop the synthesis primer, bound at the 5’ end of the primer
o Modifies nucleotide that will stop the during synthesis
synthesis  Binding shows color reaction
o Termination is a known value  Aliquots to color the primer
 Components o Dye terminator
o ssDNA template  Performed with one of the four
o M13 bacteriophage as primer fluorescent dyes attached to each of the
o Deoxynucleotides for synthesis (dNTP) ddNTP instead of the primer and labelled
o Dideoxynucleotides for termination (ddNTP) at the 3’ end
o 32P-fluorescent dye labeled nucleotide  Termination shows color reaction
 Involves aliquot of samples into four tubes with  Color in ddNTP
primer each containing dNTP for synthesizing,
one type of each ddNTP is added for every tube
to facilitate termination of the sequence
o Random adding of ddNTP
o DNA sample will undergo synthesis 
attachment of ddNTP on the sequence will
stop the sequence
Sequencing Interpretation

 Both dye primer and dye terminator sequencing

are loaded onto a slab or capillary gel
 Electropherogram
o Shows the sequence signal frequency
(requires a software)
o The quality of electropherogram depends on
the quality of the template, efficiency of the
sequencing reaction and the cleanliness of
the ladder
 Pulses means there is termination and
the highest pulse will be recorded
 ssDNA template
 Sequencing primer
 Sulfurylase and Luciferase
 Adenosine 5’ phosphate substrate
 Luciferin

 Most useful for short to moderate sequence
analysis; no need for sequencing ladder
 Relies on the luminescence when nucleotides
are added to a growing strand of DNA
 Uses: mutation and single nucleotide
polymorphism detection, HLA typing
o Primer  DNA synthesis  attach to
ddNTP (nucleotide has pyrophosphate
label)
 Detection of DNA Methylation; modification
of chain termination method
 Epigenetics: no alteration in the DNA
sequence but shows changes in phenotype
 Methylation: stops/ inactivates DNA
sequence
o Hindi matanggal yung methylation ni
cytosine

 In the presence of bisulfite, methylated cytosine

 Once a dNTP forms a phosphodiester bond with will remain as it is while the unmethylated
the primer, it releases pyrophosphate that will be cytosine will be replaced by uracil and eventually
converted to ATP by the help of sulfurylase in the changed to thymine. Cytosine remains as it is will
presence of pyrophosphate substrate. The ATP is be counted as methylated, those that are
used to generate a luminescent signal by changed to thymine will not be counted.
luciferase-catalyzed conversion of luciferin to
oxyluciferin.
 The reaction of each dNTP is added one at a time
and observed for light peak; sequences are
interpreted through pyrogram
o Every light means additions of bases
o Generate signal which is light means it binds
in the synthesis

 Recognize specific base sequences and
restricts nucleic acid polymers
 Naming system for restriction enzymes is
based on the organism they are isolated
o Came from the organism (bacteria)
often in appearance of nucleases
 Sites to be considered:
o Recognition site - recognize DNA
sequence target
o Cleavage site - active for the actual
restriction
 Classification by function:
o Endonucleases
o Exonucleases
 Internal property of enzymes.
 Discovered in early 1950s when some
bacteria are immune to bacteriophage
infection due to its additional methyl group
that prevents degradative enzyme action
o Normal bacteria infected by
bacteriophage(contain plasmids)
o Plasmids injected by bacteriophage is
not affected by the plasmids of normal
bacteria because of the addition of
methyl group to prevent infection
 Cleaves at internal phosphodiester bond of
the DNA strand
o Where DNA backbone is seen
Type I Restriction Enzyme
 Sample: EcoK1, EcoR124, EcoB
 Both nuclease and methylase activity works in
single enzyme
o Nuclease for recognition
o Methylase for cleaving or inactivation
 Recognition site: bipartite sequence separated by
6-8bp non-specific nucleotides with methylated
adenine on the sequence
o Specificity protein
o Methyltransferase protein
o Restriction endonuclease protein
 Cleavage site: 1000bp away from the recognition
site
o ATP
o S-adenosylmethionine
o Magnesium ions
 Far away from the recognition site and it’s not
use in the laboratory because it prone to error
 Non-specific cleaving
Type III Restriction Enzyme
 Sample: EcoP15, HindIII
 Both nuclease and methylase activity works in
single enzyme
 Recognition site: asymmetrical, 5-6bp in length
and requires two inversely oriented sites for
cleavage
o Methyltransferase protein
o Restriction endonuclease protein
 Cleavage site: 20-30bp away from recognition
site
o ATP
o S-adenosylmethionine (SAM)
o Magnesium ions
Type II Restriction Enzyme
 Separate action of methylase and endonuclease
activity
o More specific performance of the activity
 Uses: gene cloning, DNA fragmentation, and
analysis
o Palindromic sequence (balance or same
number of base pairs)
o Recognition and cleaving happens at the
same location
 Subtypes based on enzymatic property
o Type IIP: 90% are used, recognizes
palindromic sequence that is 4-8bp length
o Type IIS: two domains, recognizes
asymmetric but cleaves as dimers
o Type IIC: three domains, cleaves outside the
recognition sequence
o Type IIT: two catalytic enzymes resulting to
nicks
 Cleavage of the enzymes may result to (1)
overhangs (2) blunt ends
 Considerations for type II endonucleases
 o Isoschizomers
 Pair of restriction enzyme that
recognizes the same recognition
sequence and cuts at the same location
 Sph1, Bbu1
o Neoschizomers
 Restriction enzymes that recognize the
same sequence but cuts differently
 Sma1, Xma1
o Isocaudomers
 Recognizes slightly different sequences
but produces the same ends
 Sau3A, BUMHI
 Degrades DNA from the 3’ hydroxyl end or 5’
phosphate end of the strand
 Primary responsible for hydrolysis of the
terminal end of nucleic acids
 Uses: degrading excess single stranded
products, unidirectional deletions, proof
reading
Exonuclease I
 Isolated from E. coli that degrades ssDNA from
the 3’ hydroxyl end
 Optimal on long single stranded ends and slows
down towards double stranded region
Exonuclease III
 Employed to remove long single strands
protruding from dsDNA
 Relies on the restriction endonucleases able
to cleave at specific base pair sequence
 Comparative analysis of fragments
generated by cleavage with two different
restriction endonucleases enables the
determination and relative location of
recognition sequence
 Determines the sizes of DNA fragments
generated by single or combination of
restriction enzyme digestion and subsequent
construction of DNA map
Restriction Map
 Uses a known restriction sites within a sequence
of DNA
 Mapping can be either (1) linear map (2) plasmid
recombinant map
o Linear: determines linear order of restriction
sites
o Circular: determine the circular order of
restrictions sites
 Fragmentation is achieved by either (1) single
digest (2) double digest
o Single digest: uses a single type of restriction
enzyme
o Double digest: uses two different type of
restriction enzyme

 Isolated from E. coli that degrades dsDNA from

the 3’ hydroxyl end from both strands
 Transforms dsDNA to become ssDNA for dideoxy
sequencing

Exonuclease V

 Isolated from E. coli that degrades dsDNA from

both ends
 Contains 3 subunits: helicase, exonuclease and
endonuclease
Linear enzyme mapping
Exonuclease VII
 Specimen 1: no enzyme 100bp
 Isolated from E. coli that degrades ssDNA from
 Specimen 2: 2 fragments= 20bp, 80bp ( EcoR1)
both ends
 Specimen 3: 2 fragments= 35bp, 65bp (Hind III)
  Specimen 4: 3 fragments= 20bp(from EcoR1),
35bp (from Hind III), 45bp (excess) =
(Combination) 22
 One of the evidence of the activity of restriction
enzyme is having fragments

 Inserting a DNA fragment to a bacterial

plasmid vector by the aid of restriction
enzyme.
 Plasmid needs to be cut off before the DNA
fragment can be inserted to the plasmid
o Plasmid  target area (recognition site)
 cut (exposure)  add target gene to
connect to plasmid called recombinant
plasmid
o Plasmid – used to store DNA sequences
(rare cases)

Application

 DNA fingerprinting for detection of genetic

abnormalities
 DNA profiling in crime scene for forensic studies
 For bacterial plasmid recombinant studies