Molecular Biology
LECTURE | PCR TECHNIQUE
WALLACE KANLEY GAZA
AMPLIFICATION TECHNIQUE
 Method that generates enough copies of single gene
sequence.
 Increases the yield of nucleic acid sequence of
interest for subsequent analyses.
 From DNA extraction, need to amplify yung na extract
before mag proceed sa detection kasi maliit lang yung
yield nung mga DNA na nakukuha natin from nucleus, so
kailangang paramihin bago sya i-detect; like sa bacte
need muna paramihin yung colonies through bacterial
culture; PCR is like bacterial culture pianparami muna
yung sample para ma detect natin sya ng maayos.
 Other amplification methods have been developed
based on making modifications of PCR.
 The methods that have been developed to amplify
nucleic acids can be divided into three groups,
based on whether the target nucleic acid itself, a
probe specific for the target sequence, or the
signal used to detect the target nucleic acid is
amplified.
 For standard PCR procedure ang pinaparami natin is yung
target nucleic acid
 Amplification technique generates enough copies of a
single genes sequence required propagation of millions of
cells in culture or isolation of large amounts of genomic
DNA.
POLYMERIZE CHAIN REACTION
Historical Review
 First used by Kary Mullis in 1983 in California
 Former the Polymerase-catalyzed chain reaction
 Kaya sya naging chain reaction, from a single gene
sequence paparamihin ng paparamihin like a chain
reaction.
 Bakit kasama si polymerase because ang pinaka bida is si
enzyme DNA polymerase.
 First successful amplification was a short fragment
of Escherichia coli plasmid pBR322.
 First application for diagnosis is patient with sickle
cell anemia.
 Sa plasmid nandon yung DNA ng bacteria.
General Overview
 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.
 PCR is like in vivo DNA replication; it happens in vivo or
outside the body; pareho sya ng DNA replication sa loob
OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE | MOLECULAR BIOLOGY
ng katawan pero nang yayare sya sa isang machine which
is our thermocycler PCR machine
PCR COMPONENTS
1. Primers
 Sa loob ng master mix nandon lahat ng kailangan natin
para sa ating PCR.
 Directs DNA synthesis to the desired regions;
critical component as it determines the specificity
of the entire sequence to amplify.
 Chemically manufactured on a DNA synthesizer
 Chemically manufactured para makapag bind ang mga
primers dun sa target sequence; parang ito yung probes
sa hybridization technique.
 ssDNA with 20-30bp in length and in 2 sets (1)
forward primer (2) reverse primer.
 ssDNA means single stranded DNA.
 Melting temperature (Tm) determines the starting
point of the primer as it affects the binding of the
primer to the sequence
 Both our forward and reverse primer kailangan sabay
yung kanilang melting point
 Melting temp: 50-70C
 Primers are designed to contain sequences
homologous to sites flanking the region to be
analyzed.
 Kapag sinabing flanking for ex. Ito yung ating region
under investigation or target sequence; yung primer
hindi sya mag bbind, dito lang sa target sequence.
 Kapag sinabing flanking mag b-bind ang primer before pa
sa region under investigation; kaya sya tinawag na primer
dun sya mag s-start before sa region under
investigation; sya ang panimula.
2. 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 (nitrogen bases guanine
and cytosine) content and secondary structures
may encounter difficulty in amplification and may
require modifications.
 More than 65% yung guanine and cytosine content it can
increase hydrogen bonding between our DNA which
makes it resistant from melting; mas mahihirapan yung
machine na idenaturate yung dsDNA
The template may be derived from:
 The patient’s genomic or mitochondrial DNA
 Viruses, Bacteria, Fungi, or Parasites
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 3. Deoxyribonucleotide bases
 Nucleotide triphosphates are the building blocks of
DNA.
 dNTPs ang mag bibind sa ating target sequence.
 Standard procedures require 0.1–0.5 mM
concentrations of each nucleotide.
 They must be equal or equimolar; meaning kapag ang
concentration ng adenine is 0.1mM kailangan ang guanine,
thymine, cytosine ay ganun din sya
 dNTPs: deoxynucleotidetriphosphates
•Adenine – deoxyadenosinetriphosphate (dATP)
•Guanine – deoxyguanosinetriphosphate (dGTP)
•Thymine – deoxythymidinetriphosphate (dTTP)
•Cytosine – deoxycytidinetriphosphate (dCTP)
4. DNA Polymerase
Thermostable enzymes (Derived from bacteria)
•Taq polymerase from Thermus aquaticus (most famous)
•Tth polymerase from Thermus thermophilus
 This DNA polymerase could be added at the beginning of
the procedure and at the it would maintain its activity
throughout the heating and cooling process.
 Bakit kailangan thermo stable ang gamit natin for PCR?
Because our PCR machine we use different temp. on
different steps so the enzyme used must be resistant
to the cooling and heating throughout the process
 Need nyang ma withstand yung temp during the process
Stoffel fragment
•Modified Taq polymerase that lacks N-terminal 289 amino
acid
•Has broader range of magnesium chloride concentration
than Taq
•Ideal for amplifying regions with high GC content
 Ito na yung tinatawag na nating modification para sa mga
sequences na maraming guanine and cytosine content
5. DNA Polymerase
 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
 Potassium chloride (20-100mM)
 Ammonium sulfate (15-30 mM)
 Magnesium chloride (1-4 mM)
 Tris-HCl (10 mM) – pH8-9.5 for proper buffering
Accessory buffer
 Bovine serum albumin (10-100ug/mL)
 Dithiothreitol (0.01 mM)
 Formamide (1-10%)
 Chaotropic agents: triton X-100, glycerol, dimethyl
sulfoxide (1-10%)
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6. PCR Controls
 Running controls are 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.
 Known sample with a known sequence
 Kapag irurun natin yung positive control kailangan meron
sya nung known sequence na hinahanap natin para alam
natin kung tama ba or nag rurun ba ng maayos yung ating
machine
Negative control without DNA
 also called contamination control or reagent blank
 ensures that the reaction mix is not contaminated
with template DNA or amplified products from a
previous run.
 wala syang laman; reagent lang; pwedeng guamamit ng
distilled water.
Negative control with DNA
 that lacks the target sequence (negative template
control)
 ensures that the primers are not annealing to
unintended sequences of DNA.
 kapag chineck yung primer kailangan yung primer mag b-
bind lang sya dun sa target sequence.
 Dapat hindi sya mag bind sa unintended sequences of
DNA na hindi naman yun ang ating target sequence.
7. Thermal Cyclers
 Designed to ramp through the required incubation
temperatures rapidly and automatically in several
cycles.
Rapid PCR
 small sample volume
 wala syang quantity; we can only detect the quality of
sample kung present siya or hindi pero wala syang
specific number.
Real time PCR
 equipped with fluorescent detectors to measure the
product.
 na ququantify natin kung ilan yung amount ng DNA
sequence present sa ating sample
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  This is already computerized; laaht yan ay ilalagay nyo na
lang sa machine and yung machine na yung bahaalng
gumawa non basta tama yung primers and dNTPase and
optimal yung temp and pH.
 Amplification program consists of a specified
number of cycles that are divided into steps during
which the samples are held at particular
temperatures for designated times.
 The temperature will then determine the reaction
that occurs, and changing the temperature changes
the reaction.
1. Denaturation
 dsDNA is denatured into two single strands
 ineexpose natin yung DNA sa mataas na temp.
 Temperature: 90-96 C
 Time: 20-60 secs
 Optimal temp: 94-96C (the best and most
favorable result)

 So paano ba gumagaana ang ating PCR machine? So first

i-cucut muna sya sa gitna yung ating DNA sequence, so
magiging ssDNA na lang sya
 Then mag uundergo sya ng annealing; kapag sinabing
annealing i-aadd natin yung primer natin mag b-bind sya
or mag f-flank sya dun sa sequence before sa region
under investigation
 Then mag a-attach yung ating mga dNTPase sa ating
target sequence then extension
2. Annealing
 Kapag sinabing extension mag b-bind na yung ating mga
 Most critical step for the specificity of the PCR
dNTPase sa ating na hiwalay na dsDNA and mag f-from
 Sa annealing na mag bbind or mag f-flank ang ating
na sila nang dalawang complete dsDNA; hanggang s
primers; dun na tayo mag s-start gumawa ng bagong
amakabuo sila ng millions of copies
dsDNA magagawang panibagong copy ng ating DNA
 Kung hindi tama yung nadikitan nung ating primer wala
BASIC PROCEDURE
tayong
 Within one to two hours PCR can produce millions of
 Attachment of primers to the strand to be
copies called amplicons of DNA; the real advantage
amplified
of the PCR is the ability to amplify specific targets.
 Bababaan ang temp
 The product is amplicon
 Temperature: 50-70 C
 PCR presents the opportunity to amplify and
 Time: 20-90 SECS
essentially clone the target sequences.
 The amplified target, then, can be subjected to
innumerable analytical procedures.
 It can be detected using diff. method
1. Electrophoresis
2. Spectrophotometry
3. Fluorometry
4. Nano drop
5. Hybridization
 The components of the PCR, DNA template, primers,
nucleotides (dNTP’s), polymerase, and buffers, are
subjected to an amplification program.

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 3. Extension  Nasa isang master mix, pinasok sa thermal cycler,
 It is the addition also of DNA polymerase which makes nilagay yung sample mag hihintay lang ng oras, mag
it possible for our dNTPase to bind sa ating sample. papalit lang ng temp, si PCR na ang bahalang gumawa.
 DNA polymerase catalyzes the formation of
phosphodiester bond to replicate DNA by extending
the primers (in vitro DNA synthesis)
 Si DNA polymerase na yung mag didikit sa isang bagong
gawa na strand sa ating lumang DNA template.
 For every cycle of a one dsDNA, it will be replicated
into two dsDNA.
 Temperature: 68-75 C
 Time: 10-60 SECS
 Optima temp: 68-72C
 From 1 DNA template after 1 cycle meron na tayong 2
kapag nag run ulit ng another cycle yung 2 magiging 4
tapos from 8 copies to 16 to 32 hanggang 35 th cycle
makakabuo tayo ng 68billion copies, exponential yung
multiplication meeting kaya mabilis talaga sya mag
rereplicate, usually umaabot ng 35 cycles.
PCR MODIFICATION
 PCR today has been adapted for various applications;
several modifications are used in the clinical
laboratory.
 The methods that have been developed to amplify
nucleic acids can be divided into three groups:
 Within one to two hours kaya na nating makagawa ng
 Target amplification (STANDARD)
millions to billions of copies ng DNA
 Probe amplification
 In some cases, our annealing temp is close enough to
 Signal amplification
extension temp; so some procedures requires only 2
temp changes, so imbis na dalawang beses mag papalit ng Target amplification

temp ang PCR machine ginagawa na lang nilang pareho  Amplify the target nucleic acid or target sequence
yung temp ng annealing and extension and that is called There are four modification of Target amplification:
2 step PCR  Capable of detecting multiple targets in a single run
 Adenine bind to Thymine (multiplex PCR)
 Cytosine bind to Guanine  Using RNA templates (reverse transcriptase PCR)
 Amplified products as templates (nested PCR)
 Quantitating starting template (quantitative PCR,
or real-time PCR)
MULTIPLEX PCR METHOD
 More than one primer pair can be added to a PCR
for multiple amplifications are primed
simultaneously resulting in the formation of multiple
products.
 Uses: typing or identification analyses
 Example: organisms that cause sexually transmitted
diseases can be targeted in multiplex PCR using one
genital swab.
 Ex. Gustong idetetct ang HIV or Hepa B it is possible
kasi gumagamit sya ng more than 1 primers
 Pwede nyang idetect ng sabay sabay unlike in a standard
PCR procedure na isang primer lang ang pwedeng gamitin

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 NESTED PCR METHOD
 Increased sensitivity by using two pairs of primers
to amplify a single target in separate run.
 Yung na amplify na nating target I aamplify pa nya.
 The low level of target and the presence of
interfering sequences (contaminated yung na
extract) can prevent a regular PCR from working
with the reliability required for clinical applications.
 Two pairs of primers are used to amplify a single
target in two separate PCR runs. The second pair of
primers, designed to bind slightly inside of the
binding sites of the first pair, will amplify the
product of the first PCR in a second round of
amplification. The second amplification will
specifically increase the amount of the intended
product.
 Since limited lang yung sample na meron tayo, using
nested PCR dalawang PCR runs ang kaya nyang gawin, mas
maapapdami natin yung ating sample
 Also if may contamination or interfering substances kaay
nyang tanggalin yon
 Uses: specimen with limited quantity and pangit and
quality
REVERSE TRANSCRIPTASE PCR METHOD
 It is not RT-PCR
 It is use when the sample is RNA
 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.
(Complementary DNA)
 It will convert from RNA to cDNA using reverse
transcriptase
 Reverse transcriptase: enzyme that converts any
RNA sample to become dsDNA in the form of
cDNA; enzyme is inactivated at 80°C once reaction
is fulfilled.
 Kapag mag p-proceed na nang denaturation kailangan
munang inactivate yung reverse transcriptase kasi
gagamit na tayo ng ibang enzyme which is our DNA
polymerase
 Kapag na convert na ang RNA to cDNA pwede na tayong
mag proceed into a normal PCR method
 Uses: gene expression analysis.
REAL-TIME/ QUANTITATIVE PCR METHOD (RT-PCR OR
QPCR)
 Can detect in a specific value kung ilan yung target
sequence present sa ating template
 Requires dsDNA-specific dyes such as ethidium
bromide or SYBR green that is added to the sample
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before the PCR test; provides a fluorescent signal
as evidence of amplification.
 We detect the signal; mas maliwanag yung signal meaning
mas mataas or mas marami yung target sequence present
 Exponential phase: fluorescence occurs during
reaction.
Threshold cycle (C")
 Number of cycles required for fluorescent signal to
cross the threshold.
 Optimal level: based on the background or baseline
fluorescence and the peak fluorescence in the
reaction.
 Kapag titignan sya sa PCR machine, tapos meron syang
graph tapos minemessure sya sa exponential phase.
Threshold value is inversely proportional to DNA
concentration.
 Low CT value = high DNA concentration (more
starting material, few cycle need)
 High CT value = low DNA concentration (less
starting material, more cycle need)
 Uses: gold standard for SARS-Cov-2 in combination with
RT-PCR
Note:
 Sa molecular lab non tinitignan nila yung CT value or
threshold value; so may cut off yon para malaman if
positive or negative ba yung patient
 Pero ususally ang nilalabas lang naman sa result is
either positive or negative hindi na nila nilalagay
yung CT value pero yung pathologist sa molecular lab
sya na yung mag lalagay or mag iinterpret ng CT value
and mag coconfirm if positive or negative ba yung
sample
 Ang trabaho lang nating medtech is to run the test
 Ang kinaibhana nya from standard PCR and real time
PCR ay quantitative may value talaga sya unlike the
standard PCR qualitative, its either positive or
negative; hindi natin alam kung gaano kadami yung
target sequence present at the start of our
procedure
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MODX311 WEEK8: LECTURE_Midterms (M2)
MS. RUTH ELLEN T. ALONZO, RMT
OTHER AMPLIFICATION TECHNIQUES
AMPLIFICATION TECHNIQUES
Ø Target Amplification Technique
Ø Probe-based Amplification Technique
Ø Signal Amplification Technique
Ø *Ang pinagkaiba nilang tatlo is kung ano ba
yung inaamplify within the DNA template or sa
ating sample.
Ø *TARGET AMPLIFICATION TECHNIQUE –
the target itself yung inaamplify naten
PROBE-BASED AMPLIFICATION
Ø The number of target nucleic acid sequences in a
sample is not changed as it is in target
amplification procedures like PCR
Ø *Hindi kase yung target nucleic acid natin ditto
yung inaamplify naten, we use synthetic probes.
Ø Synthetic probes that are specific to the target
sequences bind to the target where the probes
themselves are amplified
Ø *Ang inaamplify natin ditto are the probe,s
which are binded to our target sequence. Its not
the target nucleic acid itself, but the probes
attached.
Ø TYPES
• Ligase chain reaction (LCR) – Abbott
• Strand displacement amplification (SDA) –
Becton Dickinson
• Qβ replicase – Vysis
LIGASE CHAIN REACTION
Ø Generates a signal by repeated ligation of probes
complementary to specific sequence
Ø Instead of DNA polymerase synthesizes new
strand by the help of primers, DNA ligase is used
to ligate the adjacent primers bound to template
forming a new DNA strand
Ø *Ligase, ligate means pagdidikitin nya.
Ø *Ano ba yung main purpose ng ating DNA ligase
doon sa DNA ligase in vivo? It closes any mix
(??) present and forms phosphodiester bonds
between existing strands.
Ø *For LCR, the ligase chain reaction generates a
signal by a repeated ligation of probes,
complementary to specific sequences in the test
DNA.
Ø *For LCR we have repeated ligation. Ulit-ulit
anf pagbbind nun gating DNA ligase into probes
complementary to our specific sequences in our
DNA template.
Ø *One complementary oligomer and one signal
producing molecule are covalently attached to
biotin for immobilization.
Ø *These two oligomer will be ligated together
only if the target is complementary.
Ø *If our probes are not complementary to our
target sequence, hindi yan icclose ni DNA ligase.
Ø *The oligomers are captured on a solid substrate
by streptavidine and it will generate a signal. If
the sequence of target is not complementary, the
captured probe will not yield a signal.
Ø *For this procedure, we have the enzymes DNA
ligase and DNA polymerase.
Ø *And because of that, it makes our LCR the
ligase chain reaction more specific and accurate
than our PCR.
Ø *For LCR process, first we have denaturation at
94 degrees Celsius, so we still have to denaturate
kasi ang sample natin ditto is still a DNA.
Kailangan muna natin paghiwalayin yung mga
strands.
Ø *Next we have Annealing, done under the
temperature 40-60 degrees Celsius, wherein
nagbbind yung apat na probes. And using DNA
ligase, pagdidikitin yang probes.
Ø *Lastly, we have Ligation, wherein our DNA
polymerase fills the gaps and the DNA ligase
closes the gaps and forms phosphodiester bonds.
Ø First application used in the detection the point of
mutation that occurs in the beta globulin of
patients with sickle cell anemia.
FARHAT, YASMINE A. 1
 MODX311 WEEK8: LECTURE_Midterms (M2)
MS. RUTH ELLEN T. ALONZO, RMT
OTHER AMPLIFICATION TECHNIQUES
STRAND DISPLACEMENT AMPLIFICATION
Ø Isothermal amplification method
Ø *Kapag sinabi nating isothermal, ibig sabihin
wala tayong temperature change dito. Kung ano
yung temperature from the start, ganun na sya
hanggang matapos yung process. Meaning hindi
natin kailangan ng thermal cycler sa ating
procedure na ito.
Ø Uses two pairs of primers and can recognize two
regions of target sequences
• Bumper primer: similar to PCR primers
• *PCR primers – they flank to our target
sequence. Kapag sinabing flank it means
nagbbind sila sa katabi na base n gating
target sequence.
• SDA primer: bound at target sequence
Ø Two-step method
• Target generation phase
• Exponential target amplification
Ø Uses: detection of M. tuberculosis, C.
trachomatis, N. gonorrhoeae
1. Target generation phase
Ø Goal: generate a new target sequence;
displaced strand used as a template
Ø *Meron tayong template, we have the SDA
primer, and the bumper primer. So sabi
natin, yung ating bumper primer it binds or
it flanks beside our target sequence. Sa
katabi nmn nya we have the primer.
Ø *Ngayon kapag ang ating SDA primer nag
extend yan, nakabuo na sya ng isang target
sequence, hahaba nmn ngayon yung ating
bumper primer. Making our SDA primer na
nag extend will be displaced.
Ø Involves denaturation of the DNA template
at 95°C; after denaturation SDA primer and
Bumper primer binds to specific sequence of
denatured DNA, new strand is formed from
the two primers thru DNA polymerase BUT
the strand produced by SDA primer will be
displaced from the template
Ø *So yung ginawa na strand n gating SDA
primer will be displaced by our bumper
primer, Yung nadismplace nay un na galling
sa SDA primer, yun yung magiging template
ulit for the new strand.
Ø *The first stage of SDA is the denaturation
of the dsDNA and annealing of primers and
probes wailed with sequences including a
restriction enzyme site.
Ø *For the second reaction, copies of the
probe incorporating the ATPAS are thereby
inactivating their restriction site on the
copying strand. The species in the target for
amplification in the second stage of the
reaction.
2. Exponential target amplification
Ø Goal: generate newer target strand
Ø Restriction enzymes creates a nick on the
target sequence, generating a substrate for
the polymerase and extends it; the displaced
strand is hybridized by a primer producing
another target sequence with hemi sensitive
restriction site
Ø *For the second phase of SDA, our target is
nick by the restriction enzyme generating a
substrate for the polymerase. So magbbind
jan yung ating DNA polymerase making
another strand of our DNA. Kasi mag
eextend yan eh, we have DNA polymerase
which extends the nick displacing the
opposite strand. So meron na nmn tayong
restriction site don na inactivate,
magkakaroon uli ng nick, magbbind
together yung DNA polymerase, maeestend
sya, madidisplace yung isang strand.
FARHAT, YASMINE A. 2
MODX311 WEEK8: LECTURE_Midterms (M2)
MS. RUTH ELLEN T. ALONZO, RMT
OTHER AMPLIFICATION TECHNIQUES
Ø *The displaced strand is hybridized by a
primer producing another endonucleolitic
target.
Ø *The product of both reactions is a copy of a
target with a hemisensitive restriction site.
Ø *So the reaction cycles of the strands are cut
and then extended. Ulit ulit sya na
magkakaroon ng nick, tapos magbbind si
DNA polymerase, madidisplace yung isang
strand, magiging template yung nadisplace
na strand for a new cycle.
Q BETA REPLICASE
Ø Named after the major enzyme used to amplify
probe sequences
Ø *Our major enzyme is the Q beta replicase which
is RNA dependent, RNA polymerase from the
bacteriophage Qbeta.
Ø *This is an enzyme that catalyzes the replication
of the RNA of bacteriophage Q beta.
Ø Target nucleic acid can either be DNA or RNA
Ø The Qβ replicase method proceeds through a
series of binding and washing steps
Ø Probe bound to the purified template is then
amplified by Qβ replicase. The resulting RNA
can be detected by fluorometry using propidium
iodide as a fluorescent label of the synthesized
probe or by chromogenic methods.
Ø Uses: detection of infectious organisms –
mycobacteria, Chlamydia, HIV,CMV
Ø *So maccapture yung probe, didikit sa magnetic
bead (?), magwwash tayo, maccapture sya sa
magneticprobe, mawwash, them marerelease
sya, then ulit ulit nag anon. Kaya series of
washing and capturing.
SIGNAL AMPLIFICATION
Ø *The number of sequences does not change. So
kaparehas lang sya n gating probe amplification.
Kung sa probe amplification ang dumadami ay
probe. Sa Signal amplification nmn ang
dumadami ay signal.
Ø Signal amplification procedures differ from
target amplification procedures in that the
number of target sequences does not change
Ø Large amounts of signal are bound to the target
sequences that are present in the sample
Ø Because the number of target sequences does not
FARHAT, YASMINE A. 3
MODX311 WEEK8: LECTURE_Midterms (M2)
MS. RUTH ELLEN T. ALONZO, RMT
OTHER AMPLIFICATION TECHNIQUES
change, signal amplification procedures are different isolates of Hepa C virus and
inherently better at quantitating the amount of HIV.
target sequences present in the clinical sample 3. By incorporating different probes that
Ø *So dahil hindi madami yung target sequences recognize slightly different sequences,
natin ditto, signal yung dumadami, it is better in multiple genotypes of the same virus can
quantitating the amount of target sequences still be detected by the same basic system.
present specially before the procedure itself. Kasi yung mga target natin ditto is more
Parang sa RT-PCR natin. of viruses and our viruses madaming
Ø Ideally performed for quantitating amount of genotypes yan.
target sequences instead of producing multiple 4. It requires multiple probes that bind to the
copies same target wwhich increases the
Ø *Usually ang goal tlga is to quantitate the target specificity of the system.
sequence present instead of producing multiple
copies.
Ø TYPES
• Branched DNA amplification (bDNA) –
Chiron Corporation
• Hybrid Capture assays – Digene Diagnostics
• Cleavage-based amplification – Third Wave
Technologies

BRANCHED DNA AMPLIFICATION

Ø Requires (1) capture probes (2) extender probes
(3) pre-amplifiers
Ø 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
Ø *Capture probes in a solid support, are
manufactured specifically to bind sa ating target
RNA or DNA making our target RNA or DNA
immobilized sa ating solid support.
Ø *Kaya sya tinawag na branched DNA, kasi yung HYBRID CAPTURE ASSAYS
mga amplifiers natin, didikit sya na parang
Ø Starts with hybridization of RNA probe to the
sanga sa ating etender probes.
denatured DNA target with hybrid-specific
Ø Uses: quantitative detection of HBV, HCV, and
immobilized antibodies; the binding of secondary
HIV-1
antibodies to RNA probes generates signal in the
Ø *There are several advantages from this method:
presence of chemiluminescent substrate
1. There is a less risk of carry over
Ø Uses: detection of HPV, HBV,CMV
contamination resulting in a positive test
Ø *It starts with hybridization of RNA probe to the
in the branched DNA assay done in PCR.
denatured DNA target. Our denatured DNA will
2. Multiple capture and extender probes can
bind to our RNA probes and will hybridize to our
be incorporated that detects slightly
antibodies present.
different targer sequence as occurs with
Ø *The RNA-DNA hybrid – is the combination of
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MS. RUTH ELLEN T. ALONZO, RMT
OTHER AMPLIFICATION TECHNIQUES
DNA and RNA probe. Is then bound by hybrid Ø *Cleavase recognizes overlap and cleavase the
specific immobilize antibodies. And a secondary signal probe which can act as an invader probe
antibody which will act as our signal bound to in the next syep of reaction.
our alkaline phosphatase which will generate a
signal in the presence of a chemiluminescent
substrate.
Ø *The captures hybrids are detected by the
binding of alkalin phosphatase conjugated anti-
DNA or RNA hybrid antibodies in a typical
sandwich assay. Kasi pumagitna yung ating
target DNA and RNA probe, yung ating DNA-
RNA hybrid na-sandwich sya sa gitna n gating
mga antibodies and the secondary antibodies
bound to our AP conjugate which will now
generate a signal.

CLEAVAGE-BASED AMPLIFIACTION
Ø Detects target nucleic acids by using a series of
probes that bind to the target and overlap
Ø Cleavase recognizes overlapping sequences of
DNA and makes a cut in the overlapping piece
Ø Uses: DNA polymorphisms, Factor V
Ø Leiden mutation detection
Ø *The target nucleic acid is mixed with the
invader and the signal probe.
Ø *Signal probe – mutant probe.
Ø *The invader and the signal probe will bind to
the target nucleic acid with the 5’ end of the
signal probe overlapping with the invader
probe.
Ø *So dahil present si enzyme cleavase, it
recognizes the overlapping sequences and
makes a cut in the overlapping piece.
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MODX311 WEEK9: LABORATORY & LECTURE_Midterms (M3)
Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
DNA SEQUENCING
Ø Sequencing methods for determining the order of
the nucleotide bases - adenine, guanine, cytosine,
and thymine - in a molecule of DNA
Ø In the clinical laboratory, DNA sequence
information is used routinely for a variety of
purposes:
• Detecting mutation
• Typing microorganisms – iba’t ibang
serotypes of viruses, tinitignan nila kung
magkakaiba ba sila ng nucleotide bases,
sequences that is one, Yung mga infectious
organisms naten just like bacteria, virus,
fungi, and parasite
• Identifying human haplotypes
• Designating polymorphisms – more on
mutations
• Targeted therapies will be directed at
abnormal DNA sequences detected by these
techniques - more on treatment
Ø METHODS
A. MANUAL SEQUENCING
• Maxam-Gilbert Method
• Sanger Method
B. AUTOMATED SEQUENCING – Eto
yung gumagamit na ng softwares,
pinakabasis nila is still sanger method. So
parang modernization lang sya nung sanger
method
C. PYROSEQUENCING – nagdedetect ng
light or luminescence
D. BISULFITE SEQUENCING – and the contents of each tube are dried and
nagdedetect naman tayo ditto ng DNA (?)
MANUAL SEQUENCING
MAXAM – GILBERT METHOD
Ø Developed by Allam Maxam and Walter Gilbert
in the year 1970s
Ø Also called “Chemical Sequencing Method”
Ø *It is called chemical sequencing method
because of the use of different chemical agents or
yung mga tinatawag nating base modifiers,
which is responsible for cleaving or breaking a
specific nucleotide.
Ø *So meron tayong nga ginagamit ditto na based
modifiers which are chemicals and they are
responsible for clearing or cleaving (?) a specific
nucleotides
Ø Requires a double- or single-stranded version of
the DNA region to be sequenced, with one end
radioactively labeled
Ø *So meron tayong ginagamit dawn a radioactive
label. It is binded at the 5’ end of our DNA
strand.
Ø *So kung maaalala nyo yung ating hybridization
based technique, meron tayo ginamit doon na
radioactive label. 32P or the radioactive
phosphorus, is actually the same label we use in
the DNA sequencing. Same with hybridization
based technique.
Ø For sequencing, the labeled fragment, or
template, is aliquoted into four tubes and each
aliquot is treated with a different chemical with
or without high salt
Ø Upon addition of a strong reducing agent, such as
10% piperidine, the single stranded DNA will
break at specific nucleotides
Ø *Apart from the base modifier, we also add a
strong reducing agent which helps in facilitating
the breakage or the cleaving of a specific
nucleotide.
Ø After the reactions, the piperidine is evaporated,
resuspended in formamide for gel loading
Ø The fragments are then separated by size on a
denaturing polyacrylamide gel
Ø *Gel electrophoresis - the method which uses
gel and electric current to separate the fragments
based of the size.
Ø The entire process lasts for 1-2 hours for short
fragments and 7-8 hours for longer fragments
Ø Components
• dsDNA or ssDNA template
• 10% piperidine as reducing agent (attaches to
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Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
5’ end)
• Base modifiers (1) dimethylsulphate (2)
formic acid (3) hydrazine (4) hydrazine with
salt
• 6% polyacrylamide gel for separation of
fragmented DNA (6-20% ideal)
• Loading dye for gel electrophoresis (such as
bromophenol blue or xylene cyanol)
*For our first tube, we have dimethylsulfate, ang
action ng dDMS is it methylates guanine. So when
methylation occurs, the chain breaks at guanine, So
ibig sabihin yung buong dna strand ay magbbreak
at mapuputol sya kapag nagkaroon ng impact ang
DMS kay guanine. Yung yellow I s the probe label
*Next is the formic acid, tinatarget nya yung
adenine and guanine (purines). It protonates
purines, making a break in our chain at guanine
and adenine .
* Hydrazine, splits pyrimidine rings, so anf target
natin ditto is si thymine and cytosine.
*It splits cytosine rings. So hihiwalay yung mga
cytosine
*First iaaliquote yung mga DNA samples natin into
4 tubes. DNA sample sa first tube, DNA sample sa
second, third, and fourth tube. And then each tube,
ang ilalagay lang natin is one specific base modifier
na nagtatarget ng specific base.
*SO first DMS sa tube 1, tube 2 formic acid, tube 3
hyddrazine, tube 4 hydrazine with salt.
Ø The denaturing conditions (formamide, urea, and
heat) prevent the single strands of DNA from
hydrogen bonding with one another or folding up
so that they migrate through the gel strictly
according to their size
Ø The sequence is inferred from the bands on the
film
Ø The smallest (fastest-migrating) band represents
the base closest to the labeled end of the
fragment
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Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
Ø The lane in which that band appears identifies the
nucleotide
Ø Sequence is read from the bottom (5′ end of the
DNA molecule) to the top (3′ end of the
molecule) of the gel
Ø *so kapag nagkaroon ng formation ng band, kasi
yung tube natin, ilalagay natin yan sa gel, at
ilalagay natin yan bawat tube sa well.
Ø INTERPRETATION:
• G + A and G – read as Guanine (G)
• G + A – read as Adenine (A)
• C + T and C – read as Cytosine (C)
• C + T – read as Thymine (T)
*Example, we have a band present at guanine and
adenine, so ano yung nucleotide present? Its
adenine.
*Meron tayong band present at cytosine and
thymine and cytosine, the nucleotide present is
cytosine.
*A band present for guanine and guanine and
adenine. The nucleotide present is guanine.
*Next meron tayong band na present sa cytosine
and thymine, the nucleoride present is thymine.
*And so on and so forth hanggang mabuo yung
ating gel. With that, nasequence natin yung DNA
molecule natin, kung pano yung pagkakasunod
sunod. Yung order ng mga nucleotides naten. That
is DNA sequencing.
*So meron tayong band present for cytosine and
cytosine + thymine. And nucleotide present natin
will be cytocine.
*A band present for Cytosine + tymune. The
nucleotide present will be Thymine
*Next we have adenine + guanine. The nucleotide
present will be adenine
*A band present for cytosine and cytosine +
thymine. The nucleotide will be cytosine
*A band present for both Adenine+guanine and
guanine. The nucleotide will be guanine
*A band present in cytosine + thymine. The
nucleotide will be thymine.
*A band present for adenine + guanine. The
nucleotide will be adenine.
*5’ – CTACGTA – 3’
PROCEDURE : MAXAM – GILBERT METHOD
1. dsDNA is denatured into ssDNA
2. Prepare four test tubes and pipette your ssDNA
sample to each test tube
3. Add the required chemical on each test tube
• Tube 1 – add Dimethylsuphate
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Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
• Tube 2 – add Formic acid
• Tube 3 – add Hydrazine
• Tube 4 – add Hydrazine with salt
4. Add the reducing agent (10% piperidine) at
90°C to facilitate breaking of the strand
5. Proceed to polyacrylamide gel electrophoresis to
separate the fragments based on their size
• Well 1 – tube 1
• Well 2 – tube 2
• Well 3 – tube 3
• Well 4 – tube 4
6. Proceed to autoradiograph to transfer the bands
to a X-ray film to be observed
7. Read the sequence starting from the bottom to
top
LIMITATIONS:MAXAM–GILBERT METHOD
Ø Although Maxam-Gilbert sequencing is a
relatively efficient way to determine short runs
of sequence data
Ø The method is not practical for high throughput
sequencing of long fragments
Ø *So okay pa sya gamitin kapag maiksi lang
yung mga fragment, pero kapag mahahaba na
di na sya ganun kapractical gamitin.
Ø In addition, the hazardous chemicals hydrazine
and piperidine require more elaborate
precautions for use and storage
Ø This method has therefore been replaced by the
dideoxy chain termination sequencing method
(or the SANGER method) for most sequencing
applications
SANGER METHOD
Ø Discovered by Fredrick Sanger in the year 1977
Ø Also known as the “Chain Termination Method”
or “Dideoxy Chain Termination Sequencing
Method” by using dideoxynucleotides (ddNTPs)
that stops DNA synthesis at specific nucleotide
Ø *Sa sanger method, gumagamit tayo ng dideoxy
nucleotides or our ddNTPs
Ø *So ano ang pinagkaiba ng ddNTPs sa dNTPs?
Ang dNTP natin meron tayong hydroxyl group,
but in ddNTPs, wala tayong hydroxyl group,
hydrogen lang sya walang oxygen. It lacks
hydroxyl group on the 3’ ribose carbon. And the
hydroxyl group is required in the formation of
the phosphodiester gland.
Ø *So ibig sabihin, kapag wala tayong hydroxyl
group sa ating ddNTPs, kapag nagbind yung
ddNTP naten sa ating template, the primer and
the DNA polymerase will not be able to extend
the template, it stops the synthesis. Yun yung
ginagawa ni ddNTP, kasi wala syang hydroxyl
group di sya makakapagform ng phosphodiester
bond.
Ø Ideally used for DNA base pairs of less than
1,000 in length giving its 99.99% base accuracy
and considered as gold standard for DNA
sequences
Ø Modification of the DNA replication process; a
short, synthetic single stranded DNA fragment
(primer) complementary to sequences just 5′ to
the region of DNA to be sequenced is used for
priming dideoxy sequencing reactions
Ø Components
• ssDNA template
• M13 bacteriophage as primer
• Deoxynucleotides for synthesis (dNTP)
• Dideoxynucleotides for termination (ddNTP)
• 32P-fluorescent dye labeled nucleotide
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MODX311 WEEK9: LABORATORY & LECTURE_Midterms (M3)
Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
PROCEDURE: SANGER METHOD
1. dsDNA is denatured into two single stranded
DNA (ssDNA)
2. 1:1 mixture of template and primer is placed
into four separate reaction tubes in sequencing
buffer
3. Sequencing buffer is usually provided with the
sequencing enzyme and contains ingredients
necessary for the polymerase activity
4. Mixtures of all four dNTPs and one of the four
ddNTPs (dATP, dGTP, dTTP, and dCTP) are
then added to each tube, with a different ddNTP
in each of the four tubes
5. With the addition of DNA polymerase enzyme
to the four tubes, the reaction begins; after about
20 minutes, the reactions are terminated by
addition of a stop buffer
• The stop buffer consists of 20 mM EDTA to
chelate cations and stop enzyme activity,
formamide to denature the products of the
synthesis reaction, and gel loading dyes
(bromophenol blue and/or xylene cyanol)
6. The sets of synthesized fragments are then
loaded onto a denaturing polyacrylamide gel
7. The products of each of the sequencing
reactions are loaded into four adjacent lanes,
labeled A, C, G, or T, corresponding to the
ddNTP in the four reaction tubes
8. Once the gel is dried and exposed to x-ray film,
the fragment patterns can be visualized from the
signal on the 32P-labeled primer (radioactive
phosphorus)
• The four-lane gel electrophoresis pattern of
the products of the four sequencing
reactions is called a sequencing ladder
• The ladder is read to deduce the DNA
sequence
• From the bottom of the gel, the smallest
(fastest migrating) fragment is the one in
which synthesis terminated closest to the
primer
• *SO 5’ end parin tayo sa baba and 3’ end
sa taas. Unlike sa ating maxam – gilbert
method, merong mga base modifiers na
specifically nagbbreak or yung action nya
is for purines and pyrimidines lang. So with
the help of our ddNTP, mas specific yung
target natin for sanger method. So pag
adenine, adenine nalang, kapag cytocine
cytosine nalang. So yung mga value natin
mas specific, direct nalang sya. SO pag
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Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
may band na present sa cytosine syempre
yung nucleotide present is cytosine. AUTOMATED FLOURESCENT
SEQUENCING
Ø Based on the principle of Sanger sequencing
Ø Uses double-stranded templates and cycle
sequencing
Ø Because cycle sequencing (unlike manual
sequencing) does not require sequential addition
of reagents to start and stop the reaction, cycle
sequencing is more easily adaptable to high
throughput applications and automation
Ø *So sa manual method kasi, nagaadd tayo ng
DNA polymerase to start the reaction, and then
nagaadd tayo ng stop buffer after 20 minutes
CLASSIC METHOD para magstop or magterminate yung reaction.
Kay automated flourescent sequencing, di na
Ø Classic method uses four test tubes that natin kailangan lagyan ng buffer di na natin
designates each ddNTPs used kailangan lagyan ng DNA polymerase. Lahat yan
automated na sa machine.
Ø Electrophoresis and reading of the sequencing
ladder can also be automated
• Use of fluorescent dyes instead of radioactive
nucleotides to label the primers or sequencing
fragments
• Fluorescent dyes used for sequencing have
distinct colors or peak wavelengths of
fluorescence emission, that can be
distinguished by automated sequencers
• Advantage of having four distinct colors is
MODERN METHOD that all four of the reaction mixes can be read
Ø Modern method uses fluorescent label to each in the same lane of a gel or on a capillary
ddNTPs and added to a single tube alone • *isang sample isang lane nalang ng gel ang
gagamitin natin instead of 4.
• *Capillary electrophoresis – instead of using
a slot of gel, ang ginagamit natin is capillary.
So sa capillary nay an sabi natin meron
tayong mg assigned colors for the HddNTP.
Isang capillary nalang sya instead of using 4
wells. Isang copy and sunod sunod na sya
magkakaiba lang ng color.

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MODX311 WEEK9: LABORATORY & LECTURE_Midterms (M3)
Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
METHODS OF SEQUENCING
DYE PRIMER
Ø Four different fluorescent dyes are attached to
four separate aliquots of the primer, bound at the
5’ end of the primer during synthesis
Ø Binding shows color reaction
Ø *Same with our manual method wherein our
radioactive label is binded at our 5’ end.
DYE TERMINATOR
Ø Performed with one of the four fluorescent dyes
attached to each of the ddNTP instead of the
primer and labelled at the 3’ end
Ø Termination shows color reaction
Ø *Hindi sa primer ang pagbbind n gating label
but in the ddNTPs
LABEL FRAGMENTS SYNTHESIZED
ACCORDING TO TERMINAL END OF THE
SAMPLE
Ø ddATP – read as A in the termination sequence
(green)
Ø ddCTP – read as C in the termination sequence
(blue)
Ø ddGTP – read as G in the termination sequence
(black/yellow)
Ø ddTTP – read as T in the termination sequence
(red)
*So ang titignan natin ngayon yung peak o yung
pinaka mataas na wavelength. So in this case
ang pinaka mataas is black, which represents
guanine.
*So for automated fluorescent sequencing ang
tinitignan nalang natin is color.
SEQUENCING INTERPRETATION
Ø Both dye primer and dye terminator sequencing
are loaded onto a slab or capillary gel
Ø Electropherogram
• Shows the sequence signal frequency (requires
a software)
• The quality of electropherogram depends on
the quality of the template, efficiency of the
sequencing reaction and the cleanliness of the
ladder
PYROSEQUENCING
Ø Most useful for short to moderate sequence
analysis; no need for sequencing ladder
Ø Relies on the luminescence when nucleotides
are added to a growing strand of DNA
Ø Uses: mutation and single nucleotide
polymorphism detection, HLA typing
Ø *HLA – The human leukocyte antigen, madalas
na tinetest ito for compatibility when it comes to
organ transplant.
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Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
Ø Components of Pyrosequencing
• ssDNA template
• Sequencing primer
• Sulfurylase and Luciferase
• Adenosine 5’ phosphate substrate
• Luciferin
Ø Relies on the generation of light (luminescence)
when nucleotides are added to a growing strand
of DNA
Ø No gels, fluorescents dyes of ddNTPs used
Ø *Ang tinitignan natin instead of dyes or bands,
it’s the light (?)
Ø Once a dNTP forms a phosphodiester bond with
the primer, it releases pyrophosphate that will be
converted to ATP by the help of sulfurylase in
the presence of pyrophosphate substrate
Ø The ATP is used to generate a luminescent
signal by luciferase-catalyzed conversion of
luciferin to oxyluciferin
Ø The reaction of each dNTP is added one at a
time and observed for light peak; sequences are
interpreted through pyrogram
Ø *So we have our DNA sample o yung ating
template, Inadd natin yung dNTPs natin and
DNA + dNTPs = pyrophosphate with the help
of DNA polymerase.
Ø *So may pyrophosphate na tayo and meron din
tayong substrate o yung ating adenine and
sulfate. Adenine and sulfate + pyrophosphate
with the help of enzyme sulfurinase, it will be BISULFATE DNA SEQUENCING
converted to ATP. And ATP with the help of Ø Detection of DNA Methylation; modification of
enzyme luciferase which is an enzyme that chain termination method
generates light in fireflies, makakapagproduce Ø *So instead na genetics ang tinitignan ditto, its
tayo ng light. And dinedetect natin sa ating epigenetics.
pyrogram is the light peaks and then each dNT, Ø Epigenetics: no alteration in the DNA sequence
they are added one at a time sa ating flow cell. but shows changes in phenotype (physical
Ø *So ang tinitignan natin kung ano yung mga characteristics of one organism)
nucleotide present kapag nakapagproduce sya Ø Methylation: addition of methyl groups to
ng light. cytosine inactivating the gene
Ø In the presence of bisulfite, methylated cytosine
will remain as it is while the unmethylated
cytosine will be replaced by uracil and eventually
changed to thymine
Ø Cytosine remains as it is will be counted as

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MODX311 WEEK9: LABORATORY & LECTURE_Midterms (M3)
Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING
methylated, those that are changed to thymine
will not be counted

*Imagine we have 2 rats a black and a white rat. So

sabi nya identical daw sila sa DNA sequence, so
pareho sila ng mga base pairs, pero bakit magkaiba
sila ng phenotype? Kasi sa white rat natin, meron
tayong tinatawag na methylation.
*Methylation – a methyl group attaches to cytosine
which inactivates the gene making a change in the
phenotype of one organism. So from black, nagging
white sya kasi may nagbind na methyl dun sa
cytosine nya.

*So sa genetics, merong alteration in DNA

sequence, si adenine nagbind sya kay cytocine,
dapat ang complementary base nya is guanine,
*Sa bisulfide sequencing. The methylated cytosine
napalitan sya ng ibang base pair, nagkaroon ng
will remain as it is whyle the unmethylated cytosine
deletion or pwedeng nareplace with other base
will be replaced by uracil and eventually change
pairs. So the alteration in DNA sequence changes
into thymine. So sa bisulfide sequencing, ang
the phenotype of one organism.
binibilang natin is kung ilan yung cytosine na
*The addition or attachment of the methyl group in
methylated.
the cytosine changes the phenotype of the organism
*So ang binibilang natin after the bisulfide
also pero walang alteration sa DNA sequence nya.
conversion kung ilan pa din yung mga cytosine
Parehas na may changes sa phenotype pero sa
present. Di natin binibilang yung mga cytosine na
genetics may alteration ng DNA sequence sa
nagging urasil and eventually magiging thymine.
Epigenetics wala.
*Ang binibilang natin is yung mga cytosine after
bisulfide conversion.

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MODX311 WEEK9: LABORATORY & LECTURE_Midterms (M3)
Ms. Ruth Ellen T. Alonzo
DNA SEQUENCING

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