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Development/Invention of PCR Technique

Kary Banks Mullis, PhD.

American biochemist, author, and lecturer.

Alma mater
Georgia Institute of Technology(BS, 1966)
University of California, Berkeley(PhD, 1973)

1993 Nobel Prize in Chemistry

Instrumentation

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Consumables

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PRINCIPLES OF DNA ISOLATION &

PURIFICATION

DNA can be isolated from any

nucleated cell.
DNA is a giant anion in solution.

Sources of DNA include

• Blood
• Buccal cells
• Cultured cells (plant and animal)
• Bacteria
• Biopsies
• Forensic samples i.e. body fluids, hair follicles, bone
& teeth roots.

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DNA isolation is a routine procedure to collect DNA for subsequent

molecular analysis. There are three basic steps in a DNA extraction:

• Cell disruption:- This is commonly achieved

by grinding or sonicating the sample.
Removing membrane lipids by adding a
detergent.
• Isolation of DNA:- Removing proteins by
adding a protease (optional but almost
always done).
• Precipitating the DNA :-usually ice-cold
ethanol or isopropanol is used. Since DNA is
insoluble in these alcohols, it will aggregate
together, giving a pellet upon centrifugation.
This step also removes alcohol soluble salt.

Basic rules
• Blood – first lyse (explode) the red blood cells with a gentle detergent
such as Triton-X-100.

• Wash cells – haemoglobin (and other pigments) inhibits restriction

enzymes and TAQ polymerase.

• Work on ice to slow down enzymatic processes.

• Wear gloves to protect your samples from you!!

• Autoclave all solutions and store in fridge (except SDS and organic
solvents!)

• Keep all pellets & supernatants until you have the DNA you want.

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Getting to the DNA

• Cells – lyse all cells in presence of :

• NaCl so that DNA is stabilised and remains as a double helix,

• EDTA which chelates Mg++ and is a co-factor of DNAse which chews up
DNA rapidly.
• anionic detergent SDS which disrupts the lipid layers, helps to
dissolve membranes & binds positive charges of chromosomal proteins
(histones) to release the DNA into the solution.
• Include a protease (proteinase K) to digest the proteins
• incubate the solution at an elevated temperature (56oC to inhibit
degradation by DNAses) for 4-24 hrs.

Getting rid of the protein

• Organic solvent extraction using equal
volume phenol:chloroform (24:1)

• Protein at the interface after centrifugation

(10000 rpm at 10o c for 10 min.)

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Precipitating the DNA

• add 2.5 - 3 volumes ice-cold 95% ethanol to the DNA
& leave at -20oC overnight.
• Centrifuge sample at 10000 rpm ,10 min., 40C.
• Wash DNA pellet to remove excess salt in 70% EtOH
and air-dry.
• Resuspend in sterile distilled water(pH7.4)
• Store at 4oC or frozen at -20oC long term.

Quantifying the DNA

• The amount of DNA can be quantified using the formula:

DNA concentration (µg/ml) = OD260 x 100 (dilution factor) x 50 µg/ml

1000
• Nucleic acids have a peak absorbance in the ultraviolet range at about 260 nm

• 1 A260 O.D. unit for dsDNA = 50 µg/ml

• 1 A260 O.D. unit for ssDNA = 33 µg/ml
• 1 A260 O.D. unit for RNA = 40 µg/ml

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DNA purity

• The purity of the DNA is reflected in the OD260:OD 280 ratio

and must be between 1.6 and 2.00.

< 1.6 – protein contaminated

> 2.0 – chloroform / phenol contaminated

• Repurify sample.

Summary
• Sample for DNA extraction
• Lysis of cells at elevated temperature +
detergent + enzyme in salt buffer
• Removal of cellular proteins
• Precipitation of nucleic acids with ethanol
• Quantitation and purity measurement of DNA

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PCR - Polymerase Chain Reaction

• PCR is an in vitro technique for the amplification of a region of DNA which lies
between two regions of known sequence.
• PCR amplification is achieved by using oligonucleotide primers.
– These are typically short, single stranded oligonucleotides which are
complementary to the outer regions of known sequence.

• The oligonucleotides serve as primers for DNA polymerase and the denatured
strands of the large DNA fragment serves as the template.
– This results in the synthesis of new DNA strands which are complementary
to the parent template strands.
– These new strands have defined 5' ends (the 5' ends of the oligonucleotide
primers), whereas the 3' ends are potentially ambiguous in length.

Polymerase Chain Reaction

PCR
• PCR allows scientists to
make many copies of a
piece of DNA.

1. Heat the DNA so it

“unzips”.

2. Add the complementary

nitrogenous bases.

3. Allow DNA to cool so the

complementary strands
can “zip” together.

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• http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

Primer selection
• Primer is an oligonucleotide sequence – will target a
specific sequence of opposite base pairing (A-T, G-C only)
of single-stranded nucleic acids

• For example, there is a

– ¼ chance (4-1) of finding an A, G, C or T in any given DNA sequence;
there is a
– 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a
– 1/256 chance of finding a given 4-base sequence.
• Thus, a sixteen base sequence will statistically be present
only once in every 416 bases (=4 294 967 296, or 4 billion):
this is about the size of the human or maize genome, and
1000x greater than the genome size of E. coli.

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Primer Specificity
• Universal – amplifies ALL bacterial DNA for
instance
• Group Specific – amplify all denitrifiers for
instance
• Specific – amplify just a given sequence

Forward and reverse primers

• If you know the sequence targeted for
amplification, you know the size which the
primers should be anealing across
• If you don’t know the sequence… What do
you get?

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DNA Polymerase
• DNA Polymerase is the enzyme responsible for copying
the sequence starting at the primer from the single DNA
strand
• Commonly use Taq, an enzyme from the
hyperthermophilic organisms Thermus aquaticus,
isolated first at a thermal spring in Yellowstone National
Park
• This enzyme is heat-tolerant
useful both because it is
thermally tolerant (survives the melting T of DNA
denaturation) which also means the process is more
specific, higher temps result in less mismatch – more
specific replication

RFLP
• Restriction Fragment Length Polymorphism
• Cutting a DNA sequence using restriction enzymes
into pieces
specific enzymes cut specific places

Starting DNA sequence:

5’-TAATTTCCGTTAGTTCAAGCGTTAGGACC
3’-ATTAAAGGCAATCAAGTTCGCAATAATGG

Enzyme X Enzyme X
5’-TTC- 5’-TTC-
3”-AAG- 3”-AAG-

5’-TAATTT 5’-CCGTTAGTT 5’-CAAGCGTTAGGACC

3’-ATTAAA 3’-GGCAATCAA 3’-GTTCGCAATAATGG

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RFLP
• DNA can be processed by RFLP either directly (if you
can get enough DNA from an environment) or from
PCR product
• T-RFLP (terminal-RFLP) is in most respects identical
except for a marker on the end of the enzyme
• Works as fingerprinting technique because different
organisms with different DNA sequences will have
different lengths of DNA between identical units
targeted by the restriction enzymes
– specificity can again be manipulated with PCR primers

Liu et al. (1997) Appl Environ Microbiol 63:4516-4522

Electrophoresis
• Fragmentation products of differing length are
separated – often on an agarose gel bed by
electrophoresis, or using a capilarry
electrophoretic separation

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Gel Electrophoresis

• This technology allows

scientists to identify
someone’s DNA!

Steps Involved in Gel Electrophoresis

1. “Cut” DNA sample with
restriction enzymes.

2. Run the DNA fragments through

a gel.

3. Bands will form in the gel.

4. Everyone’s DNA bands are unique

and can be used to identify a
person.

5. DNA bands are like “genetic

fingerprints”.

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Macromolecule blotting & probing

Southern blotting

• Southern blot is a method for probing for the presence of a specific

DNA sequence within a DNA sample.
• DNA samples are separated by gel electrophoresis and then
transferred to a membrane by blotting via capillary action.
• The membrane is then exposed to a labeled DNA probe that has a
complement base sequence to the sequence on the DNA of
interest.
• less commonly used due to the capacity of other techniques, such
as PCR.
• Southern blotting are still used for some applications such as
measuring transgene copy number in transgenic mice, or in the
engineering of gene knockout embryonic stem cell lines.

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Northern blotting
• The northern blot is used to study the expression patterns of a specific
type of RNA molecule as relative comparison among a set of different
samples of RNA.
• RNA is separated based on size and is then transferred to a membrane
then probed with a labeled complement of a sequence of interest.
• The results may be visualized through a variety of ways depending on the
label used. Most result in the revelation of bands representing the sizes of
the RNA detected in sample.
• The intensity of these bands is related to the amount of the target RNA in
the samples analyzed.
• It is used to study when and how much gene expression is occurring by
measuring how much of that RNA is present in different samples.
• one of the most basic tools for determining at what time, and under what
conditions, certain genes are expressed in living tissues.

Western blotting
• In western blotting, proteins are first separated by size, in a thin gel sandwiched
between two glass plates in a technique known as SDS-PAGE sodium dodecyl
sulphate polyacrylamide gel electrophoresis.

• The proteins in the gel are then transferred to a nitrocellulose, nylon or other
support membrane.

• This membrane probed with solutions of antibodies. Antibodies specifically bind to

the protein of interest & visualized by a variety of techniques, including colored
products, chemiluminescence, or autoradiography.

• Antibodies are labeled with enzymes. When a chemiluminescent substrate is

exposed to the enzyme it allows detection.

• Using western blotting techniques allows not only detection but also quantitative
analysis.

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Molecular markers
• Molecular marker are based on naturally
occurring polymorphism in DNA sequence(i.e.
base pair deletion, substitution ,addition or
patterns).
• Genetic markers are sequences of DNA which
have been traced to specific locations on the
chromosomes and associated with particular
traits.
• It can be described as a variation that can be
observed.
• A genetic marker may be a short DNA
sequence, such as a sequence surrounding a
single base-pair change (single nucleotide
polymorphism, SNP), or a long one, like mini
satellites.

Some commonly used types of genetic markers are

• RFLP (or Restriction fragment length polymorphism)

• AFLP (or Amplified fragment length polymorphism)
• RAPD (or Random amplification of polymorphic DNA)
• VNTR (or Variable number tandem repeat)
• Micro satellite polymorphism, SSR (or Simple sequence
repeat)
• SNP (or Single nucleotide polymorphism)
• STR (or Short tandem repeat)
• SFP (or Single feature polymorphism)
• DArT (or Diversity Arrays Technology)
• RAD markers (or Restriction site associated DNA markers)

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There are 5 conditions that characterize a

suitable molecular marker
• Must be polymorphic
• Co-dominant inheritance
• Randomly and frequently distributed
throughout the genome
• Easy and cheap to detect
• Reproducible

Molecular markers can be used for several

different applications including
• Germplasm characterization,
• Genetic diagnostics,
• Characterization of transformants,
• Study of genome
• Organization and phylogenic analysis.
• Paternity testing and the investigation of crimes.
• Measure the genomic response to selection in
livestock

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RFLP (Restriction fragment length polymorphism)

RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which

can recognize and cut DNA wherever a specific short sequence occurs. A
RFLP occurs when the length of a detected fragment varies between
individuals and can be used in genetic analysis.

Advantages:
• Variant are co dominant
• Measure variation at the level of DNA sequence, not protein sequence.
Disadvantage:
• Requires relatively large amount of DNA

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AFLP ( Amplified fragment length polymorphism)

In this analysis we can amplify restricted fragments and reduces the
complexity of material to be analyzed (approx 1000 folds).it can be
used for comparison b/w closely related species only.
Advantages:
• Fast
• Relatively inexpensive
• Highly variable
Disadvantage:
• Markers are dominant
• Presence of a band could mean the individual is either homozygous
or heterozygous for the Sequence - can’t tell which?

RAPD ( Random amplification of polymorphic DNA)

Random Amplification of Polymorphic DNA. It is a type of PCR

reaction, but the segments of DNA that are amplified are random.
Advantages:
• Fast
• Relatively inexpensive
• Highly variable
Disadvantage:
• Markers are dominant
• Presence of a band could mean the individual is either
homozygous or heterozygous for the Sequence - can’t tell which?
• Data analysis more complicated

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Micro satellite polymorphism, SSR or Simple

sequence repeat
Microsatellites, Simple Sequence Repeats (SSRs), or Short
Tandem Repeats (STRs), are repeating sequences of 1-6 base
pairs of DNA.
Advantages:
• Highly variable
• Fast evolving
• Co dominant
Disadvantage:
• Relatively expensive and time consuming to develop

SNP
• A single-nucleotide polymorphism
(SNP, pronounced snip) is a DNA
sequence variation occurring when
a single nucleotide — A, T,C, or G
— in the genome (or other shared
sequence) differs between
members of a species or paired
chromosomes in an individual.
• Used in biomedical research ,crop
and livestock breeding programs.

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STR
• A short tandem repeat (STR) in DNA occurs when a
pattern of two or more nucleotides are repeated and the
repeated sequences are directly adjacent to each other.
• The pattern can range in length from 2 to 16 base pairs
(bp) (for example (CATG)n in a genomic region) and is
typically in the non-coding intron region
• Used in forensic cases.
• used for the genetic fingerprinting of individuals

DGGE
• Denaturing gradient gel electrophoresis
– The hydrogen bonds formed between complimentary base pairs, GC
rich regions ‘melt’ (melting=strand separation or denaturation) at
higher temperatures than regions that are AT rich.
• When DNA separated by electrophoresis through a gradient of
increasing chemical denaturant (usually formamide and urea), the
mobility of the molecule is retarded at the concentration at which the
DNA strands of low melt domain dissociate.
– The branched structure of the single stranded moiety of the
molecule becomes entangled in the gel matrix and no further
movement occurs.
– Complete strand separation is prevented by the presence of a high
melting domain, which is usually artificially created at one end of
the molecule by incorporation of a GC clamp. This is accomplished
during PCR amplification using a PCR primer with a 5' tail consisting
of a sequence of 40 GC.

Run DGGE animation here – from http://www.charite.de/bioinf/tgge/

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RFLP vs. DGGE

RFLP DGGE
• Advantages • Advantages
– Relatively easy to do – Very sensitive to variations in
– Results can be banked for future DNA sequence
comparisons – Can excise and sequence DNA in
• Limitations bands
– Less sensitive phylogenetic • Limitations
resolution than sequencing – Somewhat difficult
– Each fragment length can – ”One band-one species” isn’t
potentially represent a diversity of always true
microorganisms – Cannot compare bands
– Cannot directly sequence between gels
restriction fragments,making – Only works well with short
identification indirect fragments (<500 bp), thus
limiting phylogenetic
characterization

FISH
• Fluorescent in-situ hybridization
– Design a probe consisting of an oligonucleotide
sequence and a tag
– Degree of specificity is variable!
– Hybridize that oligonucleotide sequence to the
rRNA of an organism – this is temperature and salt
content sensitive
– Image using epiflourescence, laser excitation
confocal microscopy
• Technique DIRECTLY images active organisms
in a sample

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Fluorescentininsitu
sitehybridisation
hybridization
(FISH) using DNA probes
Probe Fluorescein
(- 20 bases) TAGCTGGCAG T
C GUAUCGACC GUC A
UA
16S rRNA
DNA

*
16S gene
* * * *
*
* *

*
*
*

*
*
* *
16S gene * * Cell
membrane

B Drift Slime Streamer

10 µm

DAPI FER656

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Oligunucleotide design

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FISH variations
• FISH-CARD – instead of a fluorescent probe on
oligo sequence, but another molecule that can
then bond to many fluorescent probes –
better signal-to-noise ratio
• FISH-RING – design of oligo sequence to
specific genes – image all organisms with DSR
gene or nifH for example

Clone Library

• http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

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http://www.ifa.hawaii.edu/UHNAI/NAIweb/presentations/astrobiol6.pdf

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Quantifying DNA/RNA by qPCR

What is qPCR
• “quantitative Polymerase Chain Reaction”
• A method that allows to follow in real time
(that is why is also called Real-Time PCR) the
amplification of a target.
• The target can be nucleic acids (RNA or DNA).
• Taq polymerase can only synthesize DNA, so
how do we study RNA using qPCR?

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Reverse Transcription
• mRNA can be copied to
complementary DNA
sequence (cDNA) using
reverse transcriptase—a
DNA polymerase that
uses ssRNA as template.
• Processed mRNA will
match protein coding
sequence while
unprocessed (nuclear)
mRNA will contain
intron sequences.

Uses of qPCR
• Precise quantitation of DNA or RNA in samples
• Estimation of gene number
• Gene expression studies by quantification of
messenger RNA

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Central Dogma of Molecular Biology:

From DNA to RNA to protein

Principle of gene expression:

From DNA to RNA to protein

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What are we doing today?

• We are using qPCR as a way to study RNA, in
this particular case messenger RNA (mRNA).
• We will test the gene expression response of a
plant to a geminivirus infection.
• This can be applied to any gene expression
analysis.

Reminder:
PCR

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Publications on qPCR have grown a lot

and its graph looks like a qPCR
amplification

Uses of qPCR
• Precise quantitation of DNA or RNA in samples
• Estimation of gene number
• Gene expression studies by quantification of
messenger RNA
– Northern blot (difficult, tedious, prone to error)
– With qPCR you can have a quick, very accurate
result (if all your controls are working)

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Different chemistries involved to

obtain quantitative signal

qPCR thermalcyclers detect

fluorescence
energy

FRET
acceptor

fluorophore fluorophore FRET donor

+ quencher
in proximity

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TaqMan

Molecular beacons

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Molecular probes

SYBRGreen

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We are using relative expression

analysis but absolute expression
analysis is possible with qPCR

ΔΔCt method
test reference

-[(Ctinf-Ctmock)-(Ctinf-Ctmock)]
2

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Cabbage Leaf Curl Virus (CaLCuV)

• Using qPCR to detect changes in
gene expression.
– Mock tissue (uninfected)
– Infected tissue
• Looking at genes involved in
Arabidopsis thaliana
pathogen response.
• cDNA prepared from RNA purified
from Arabidopsis plants
– Reverse transcription using polyT
primer and NTPs.
Arabidopsis infected with CaLCuV

Pathogen response in arabidopsis

Salicylic acid Jasmonic acid Ethylene

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Genes of interest for today

• PR1- Pathogenesis-related
• NPR1- Not expressing PR-1
• WRKY53- Transcription factor involved in pathogen response
• FAD8- Fatty-acid desaturase, involved in the production of
jasmonate (pathogen response)
• Invertase- Enzyme known to be involved in the pathogen response
• Catalase- Enzyme known to be involved in the pathogen response
• CAB-AB- Protein that takes part in the Chlorophyll complex
• UCE- Ubiquitin conjugating enzyme. Control gene. It has been
reported it doesn’t change during infection.

Prior to doing the qPCR

• Total RNA from mock and infected plants was
purified
• It was treated with DNAase to get rid of all
genomic DNA (will also amplify with primers)
• cDNA was produced and diluted to 50ng/µl

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Preparing a qPCR reaction

• Forward and reverse primers (oligos) for each
gene of interest
• cDNA of control and treated samples (mock
and infected, respectively)
• SYBRGreen PCR mix
• Thermocycler with fluorescence measuring
capabilities

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