Molecular Techniques
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Transcript of Molecular Techniques
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Reminder: All molecular techniques are based
on the chemical “personality” (or chemical properties) of the DNA molecule (or nucleic
acids)
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Cellular level
Organelle level
Molecular level: Macromolecules
Atomic level
C, H, O, N, S, P
Microscope
Cell fractionation-Nucleus-Mitochondria-Ceell membrane-Cytosol
Proteins Carbohydrates Lipids Nucleic acids
Studies of cell-Fractionation-Purification/ Identification-Structure/ Function
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Negatively-charged phosphate-sugar backbone
-- -
-
Hydrogen bonds
Specificity of nucleotides
Various lengths
Nucleic Acids
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CONTENTSRestriction Enzymes
ElectrophoresisBlotting and HybridizationPolymerase Chain Reaction
DNA Sequences
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Restriction EnzymesRestriction Enzymes
Molecular scissors which isolated from bacteria where they are used as Bacterial defense against viruses
Molecular scalpels to cut DNA in a precise and predictable manner
Enzyme produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction
sites, and then cleave both DNA strands at this site
A class of endo-nucleases that cleavage DNA after recognizing a specific sequence
Members of the class of nucleases
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Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and
RNA molecules
EndonucleaseCleave nucleic acids at internal position
ExonucleaseProgressively digest from the ends of the nucleic acid molecules
Nuclease
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Endonuclease
Type Characteristics
I Have both restriction and modification activity Cut at sites 1000 nucleotides or more away from
recognition site ATP is required
II It has only restriction site activity Its cut is predictable and consistent manner at a
site within or adjacent to restriction site It require only magnesium ion as cofactor
III Have both restriction and modification activityCut at sites closed to recognition site ATP is required
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There are already more than 1200 type II enzymes isolated from prokaryotic organism
They recognize more than 130 different nucleotide sequence
They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence
Palindromic sequence:The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand
To Avoid confusion, restriction endo-nucleases are named according to the following nomenclature
Restriction Enzymes
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The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated
The second and third letters are usually the initial letters of the organisms species name. It is written in italic
A fourth letter, if any, indicates a particular strain organism
Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery
Nomenclature
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NomenclatureEcoEcoRIRI E : Genus EscherichiaE : Genus Escherichia
co: Species colico: Species coli
R : Strain RY13R : Strain RY13
I : First endonuclease isolatedI : First endonuclease isolated
BamBamHIHI B : Genus BacillusB : Genus Bacillus
am: species amyloliquefaciensam: species amyloliquefaciens
H : Strain HH : Strain H
I : First endonuclease isolatedI : First endonuclease isolated
HinHindIIIdIII H : Genus HaemophilusH : Genus Haemophilus
in : species influenzaein : species influenzae
d : strain Rdd : strain Rd
III : Third endonuclease isolatedIII : Third endonuclease isolated
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SpecificityEnzymeEnzyme SourceSource SequenceSequence EndEnd
BamHIBamHI Bacillus Bacillus amyloliquefaciens Hamyloliquefaciens H
GGGATCCGATCC StickStickyy
BglIIBglII Bacillus globigiiBacillus globigii AAGATCTGATCT StickStickyy
EcoRIEcoRI Escherichia coli RY13Escherichia coli RY13 GGAATTCAATTC StickStickyy
EcoRIIEcoRII Escherichia coli R245Escherichia coli R245 CCTGGCCTGG StickStickyy
HaeIIIHaeIII Haemophilus aegyptiusHaemophilus aegyptius GGGGCCCC BluntBlunt
HindIIHindII Haemophilus influenzae Haemophilus influenzae RdRd
GTPyGTPyPuACPuAC BluntBlunt
HindIIIHindIII Haemophilus influenzae Haemophilus influenzae RdRd
AAAGCTTAGCTT StickStickyy
HpaIIHpaII Haemophilus Haemophilus parainfluenzaeparainfluenzae
CCCGGCGG StickStickyy
NotINotI Nocardia otitidis-Nocardia otitidis-caviarumcaviarum
GCGCGGCCGGGCCGCC
StickStickyy
PstIPstI Providencia stuartii 164Providencia stuartii 164 CTGCACTGCAGG StickStickyy
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Restriction Product
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Restriction enzymesRestriction enzymes
degenerate or specific sequences
kind of ends produced (5’ or 3’ overhang (cohesive=sticky), blunt=flush)
number of nucleotides recognized (4, 6,8 base-cutters most common)
whether cleavage occurs within the recognition sequence
Restriction enzymes can be grouped by:
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A restriction enzyme (A restriction enzyme (EcoEcoRI)RI)
1. 6-base cutter
4. produces a 5’ overhang (sticky end)
2. Specific palindromic sequence (5’GAATTC) 3. Cuts within the recognition sequence (type II enzyme)
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GEL ELECTROPHORESIS
1. For separating disperse charged biological molecule of any size/length
2. Uses electricity3. Uses a matrix 4. Uses buffer solution
First observed by Reuss, 1807
The motion of disperse charged particle relatives to a fluid under the influence of a spatially uniform electric field
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Factors affecting the mobility of molecules:
1. Molecular factors• Charge• Size• Shape
2. Environment factors• Electric field strength• Matrix (pore: sieving effect)• Running buffer
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+
Electrophoresis
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Electrophoresis
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Paper Agarose
1. purified large MW polysaccharide (from agar)2. very open (large pore) gel3. used frequently for large DNA molecules
Acrylamide 1. a white odorless crystalline solid chemical compound2. soluble in water, ethanol, ether, chloroform3. used to synthesize poly-acrylamide which find many uses as water soluble thickeners
Starch Cellulose acetate
Types of matrix (supporting media)
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DNA Agarose GelAn analytical technique used to separate DNA
by size1. Electric field induces
DNA to migrate toward the anode due to the net negative charge of the sugar phosphate backbone of the DNA
2. Longer molecules migrate more slowly
3. Visualized using a fluorescence dye special for DNA such as ethidium bromide
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acrylamide polymer very stable gel can be made at a wide variety of concentrations large variety of pore sizes (powerful sieving
effect)
Polyacrylamide Gels
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Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH3(CH2)11SO3
- Na+
Amphipathic molecule
Strong detergent to denature proteins
Binding ratio: 1.4 g SDS/g protein
Charge and shape normalization
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
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- Separate molecules according to their isoelectric point (pI)
- At isoelectric point (pI) molecule has no charge (q=0), hence molecule ceases
- pH gradient medium
Isoelectric Focusing Electrophoresis (IFE)
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- First dimension is IFE (separated by charge)
- Second dimension is SDS-PAGE (separated by size)
- So called 2D-PAGE
- High throughput electrophoresis, high resolution
2-dimensional Gel Electrophoresis
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2-dimensional Gel Electrophoresis
Spot coordination pH MW
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Blotting and HybridizationBlotting and Hybridization
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BlottingBlottingTransfer the DNA from the gel to a
solid support.
Transferring of DNA, RNA, Protein to an immobilizing binding matrix such as
nitrocellulose paper or nylon
Southern blot DNA
Northern blot RNA
Western blot Protein
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SOUTHERN BLOTTING The technique was developed by E.M. Southern
in 1975. The Southern blot is used to detect the presence
of a particular piece of DNA in a sample. The DNA detected can be a single gene, or it
can be part of a larger piece of DNA such as a viral genome
The key to this method is hybridization. Hybridization-process of forming a double-
stranded DNA molecule between a single-stranded DNA probe and a single-stranded
target patient DNA.
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SOUTHERN BLOTTING
There are 2 important features of hybridization:
• The reactions are specificThe probes will only bind to targets with a complementary sequence.
• The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.
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Southerns Blotting (DNA Blotting)
DNA fragments created by restriction digestion are separated on an agarose gel
Separated fragments are denatured and transferred to a membrane (blot) by blotting
Probe is hybridized to complementary sequences on the blot and excess probe is washed away
Location of probe is determined by detection method (e.g., film, fluorometer)
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Southern blotting
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Some Applications of DNA Blots
Map restrictions sites near a particular locus for gene isolation or allele analysis (e.g., RFLP
restriction fragment length polymorphism) Identity of closely related genes
Confirmation of gene transfer or gene disruption
Detection of foreign DNA
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RNA Blotting (Northern)
RNA is separated by size on a denaturing RNA is separated by size on a denaturing agarose gel and then transferred onto a agarose gel and then transferred onto a
membrane (blot)membrane (blot) Probe is hybridized to complementary Probe is hybridized to complementary sequences on the blot and excess probe is sequences on the blot and excess probe is
washed awaywashed away Location of probe is determined by Location of probe is determined by
detection method (e.g., film, fluorometerdetection method (e.g., film, fluorometer))
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Applications of RNA Blots
Detect the expression level and transcript Detect the expression level and transcript size of a specific gene in a specific tissue size of a specific gene in a specific tissue
or at a specific time. Sometimes mutations or at a specific time. Sometimes mutations do not affect coding regions but do not affect coding regions but
transcriptional regulatory sequences (e.g., transcriptional regulatory sequences (e.g., UAS/URS, promoter, splice sites, copy UAS/URS, promoter, splice sites, copy
number, transcript stability, etc.)number, transcript stability, etc.)
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Western BlotWestern Blot Protein blottingProtein blotting Highly specific qualitative testHighly specific qualitative test Can determine if above or below thresholdCan determine if above or below threshold Typically used for researchTypically used for research Use denaturing SDS-PAGEUse denaturing SDS-PAGE
Solubilizes, removes aggregates & adventitious Solubilizes, removes aggregates & adventitious proteins are eliminatedproteins are eliminated
Components of the gel are then transferred to a solid support or transfer membrane
Paper towel
Transfer membrane
Wet filter paper
Paper towelweight
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Western BlotWestern Blot
Add monoclonal antibodies
Rinse again
Antibodies will bind to specified protein
Stain the bound antibody for colour development
It should look like the gel you started with if a positive reaction occurred
Block membrane e.g. dried nonfat milkBlock membrane e.g. dried nonfat milk
Rinse with ddH2O
Add antibody against yours with a marker (becomes the antigen)
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HybridizationHybridization Pairing of complementary DNA and/or RNA
and/or protein
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HybridizationHybridization It can be DNA:DNA, DNA:RNA, or RNA:RNA
(RNA is easily degraded) It depended on the extent of complementation It depended on temperature, salt
concentration, and solvents Small changes in the above factors can be used
to discriminate between different sequences (e.g. small mutations can be detected)
Probes can be labeled with radioactivity, fluorescent dyes, enzymes.
Probes can be isolated or synthesized sequences
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In-situ hybridisation
Chromosome in-situ hybridisation DNA probe detects sequences in
chromosomes Map gene sequences
Tissue in-situ hybridisation RNA probe detects sequences in cells and
tissues Identify sites of gene expression Analyse tissue distribution of expression
Hybridization which is performed by denaturing the DNA of cell squash on a microscope slide so that reaction is possible with an added of probe
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Oligonucleotide probes Single stranded DNA (usually 15-40 bp) Degenerate oligonucleotide probes can be
used to identify genes encoding characterized proteins• Use amino acid sequence to predict
possible DNA sequences• Hybridize with a combination of probes• TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) -
could be used for FWMDC amino acid sequence
Can specifically detect single nucleotide changes
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Detection of Probes
Probes can be labeled with radioactivity, Probes can be labeled with radioactivity, fluorescent dyes, enzymes.fluorescent dyes, enzymes.
Radioactivity is often detected by X-ray Radioactivity is often detected by X-ray film (autoradiography)film (autoradiography)
Fluorescent dyes can be detected by Fluorescent dyes can be detected by fluorometers, scannersfluorometers, scanners
Enzymatic activities are often detected by Enzymatic activities are often detected by the production of dyes or light (x-ray film)the production of dyes or light (x-ray film)
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Polymerase Chain ReactionPolymerase Chain Reaction (PCR)(PCR)
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Polymerase Chain Reaction
Powerful technique for amplifying DNA
Amplified DNA are then separated by gel electrophoresis
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A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of
DNA/RNA from an initial complex source of nucleic acid - generates sufficient for subsequent analysis
and/or manipulationAmplification of a small amount of DNA using specific
DNA primers (a common method of creating copies of specific fragments of DNA)
DNA fragments are synthesized in vitro by repeated reactions of DNA synthesis (It rapidly amplifies a single
DNA molecule into many billions of molecules) In one application of the technology, small samples of DNA, such as those found in a strand of hair at a crime
scene, can produce sufficient copies to carry out forensic tests.
Each cycle the amount of DNA doubles
PCR
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The Ability to generate identical high copy number DNAs made possible in the 1970s by recombinant
DNA technology (i.e., cloning). Cloning DNA is time consuming and expensive
Probing libraries can be like hunting for a needle in a haystack.
Requires only simple, inexpensive ingredients and a couple hours
PCR, “discovered” in 1983 by Kary Mullis, Nobel Prize for Chemistry (1993).
It can be performed by hand or in a machine called a thermal cycler.
Background on PCR
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Three StepsThree Steps
SeparationDouble Stranded DNA is denatured by heat into single strands. Short Primers for DNA replication are added to the mixture.
PrimingDNA polymerase catalyzes the production of complementary new strands.
CopyingThe process is repeated for each new strand createdAll three steps are carried out in the same vial but at different temperatures
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Step 1: SeparationStep 1: Separation Combine Target Sequence, DNA primers
template, dNTPs, Taq Polymerase Target Sequence
1. Usually fewer than 3000 bp 2. Identified by a specific pair of DNA primers- usually
oligonucleotides that are about 20 nucleotides Heat to 95°C to separate strands (for 0.5-2
minutes)• Longer times increase denaturation but decrease enzyme and
template
Magnesium as a CofactorMagnesium as a Cofactor Mg stabilizes the reaction between:
•oligonucleotides and template DNA•DNA Polymerase and template DNA
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Heat Denatures DNA by uncoiling the Double Helix strands.
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Step 2: PrimingStep 2: Priming Decrease temperature by 15-25 °C
Primers anneal to the end of the strand 0.5-2 minutes
Shorter time increases specificity but decreases yield Requires knowledge of the base sequences of the 3’ -
end
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Selecting a PrimerSelecting a Primer
Primer length Primer length Melting Temperature (Melting Temperature (TTmm) )
Specificity Specificity Complementary Primer Sequences Complementary Primer Sequences G/C content and Polypyrimidine (T, G/C content and Polypyrimidine (T,
C) or polypurine (A, G) stretches C) or polypurine (A, G) stretches 3’-end Sequence 3’-end Sequence
Single-stranded DNASingle-stranded DNA
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Step 3: PolymerizationStep 3: Polymerization
Since the Taq polymerase works Since the Taq polymerase works best at around 75 ° C (the best at around 75 ° C (the temperature of the hot springs temperature of the hot springs where the bacterium was where the bacterium was discovered), the temperature of the discovered), the temperature of the vial is raised to 72-75 °Cvial is raised to 72-75 °C
The DNA polymerase recognizes The DNA polymerase recognizes the primer and makes a the primer and makes a complementary copy of the complementary copy of the template which is now single template which is now single stranded.stranded.
Approximately 150 nucleotides/secApproximately 150 nucleotides/sec
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Potential Problems with TaqPotential Problems with Taq
Lack of proof-reading of newly synthesized DNA.Lack of proof-reading of newly synthesized DNA. Potentially can include di-Nucleotriphosphates Potentially can include di-Nucleotriphosphates
(dNTPs) that are not complementary to the (dNTPs) that are not complementary to the original strand. original strand.
Errors in coding resultErrors in coding result Recently discovered thermostable DNA Recently discovered thermostable DNA
polymerases, polymerases, Tth Tth and and PfuPfu, are less efficient, yet , are less efficient, yet highly accuratehighly accurate..
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1.Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence.
2.Next, denature the DNA at 94˚C.3.Rapidly cool the DNA (37-65˚C) and anneal
primers to complementary s.s. sequences flanking the target DNA.
4.Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus).
5.Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA.
6.Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely.
7. Cool to 4˚C and store or use amplified PCR product for analysis.
How PCR works
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Step 1 7 min at 94˚C Initial DenatureStep 2 45 cycles of:
20 sec at 94˚C Denature20 sec at 64˚C Anneal 1 min at 72˚C Extension
Step 3 7 min at 72˚C Final ExtensionStep 4 Infinite hold at 4˚C Storage
Thermal cycler protocol Example
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The Polymerase Chain Reaction
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PCR amplificationPCR amplification
Each cycle the oligo-nucleotide primers bind most all templates due to the high
primer concentration The generation of mg quantities of DNA
can be achieved in ~30 cycles (~ 4 hrs)
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Starting nucleic acid - DNA/RNA
Thermo-stable DNA polymerase e.g. Taq polymerase
Oligonucleotides (primer) Design them well!
Buffer Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
OPTIMISING PCR
THE REACTION COMPONENTS
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Organims, Organ, Tissue, cells (hair root, callus, leaves, root, seed)
Obtain the best starting material.
Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood
Good quality genomic DNA if possible
Empirically determine the amount to add
RAW MATERIAL
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Number of options available
Taq polymerasePfu polymeraseTth polymerase
How big is the product?
100bp 40-50kb
What is end purpose of PCR?1. Sequencing - mutation detection-. Need high fidelity polymerase
-. integral 3’ ________ 5' proofreading exonuclease activity
2. Cloning
3. Marker development
POLYMERASE
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Length ~ 10-30 nucleotides (21 nucleotides for gene isolation)
Base composition:
50 - 60% GC rich, pairs should have equivalent Tms
Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]
Initial use Tm–5°C
Avoid internal hairpin structuresNo secondary structure
Avoid a T at the 3’ end
Avoid overlapping 3’ ends – will form primer dimers
Can modify 5’ ends to add restriction sites
PRIMER DESIGN
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PRIMER DESIGN
Use specific programs
OLIGOMedprobe
PRIMERDESIGNERSci. Ed software
Also available on the internethttp://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html
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Mg2+ CONCENTRATION
1 1.5 2 2.5 3 3.5 4 mM
Normally, 1.5mM MgCl2 is optimal
Best supplied as separate tube
Always vortex thawed MgCl2
Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides
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USE MASTERMIXES WHERE POSSIBLE
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How Powerful is PCR?How Powerful is PCR?
PCR can amplify a usable amount of DNA PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 (visible by gel electrophoresis) in ~2
hours.hours. The template DNA need not be highly The template DNA need not be highly
purified — a boiled bacterial colony.purified — a boiled bacterial colony. The PCR product can be digested with The PCR product can be digested with restriction enzymes, sequenced or cloned.restriction enzymes, sequenced or cloned.
PCR can amplify a single DNA molecule, PCR can amplify a single DNA molecule, e.g.e.g. from a single sperm. from a single sperm.
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Applications of PCRApplications of PCR Amplify specific DNA sequences (genomic DNA, cDNA, Amplify specific DNA sequences (genomic DNA, cDNA,
recombinant DNA, etc.) for analysisrecombinant DNA, etc.) for analysis
1. Gene isolation1. Gene isolation
2. Fingerprint development2. Fingerprint development Introduce sequence changes at the ends of fragmentsIntroduce sequence changes at the ends of fragments Rapidly detect differences in DNA sequences (e.g., Rapidly detect differences in DNA sequences (e.g.,
length) for identifying diseases or individualslength) for identifying diseases or individuals Identify and isolate genes using degenerate Identify and isolate genes using degenerate
oligonucleotide primersoligonucleotide primers• Design mixture of primers to bind DNA encoding Design mixture of primers to bind DNA encoding
conserved protein motifsconserved protein motifs
Genetic diagnosis - Mutation detectionGenetic diagnosis - Mutation detectionThe basis for many techniques to detect gene The basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10mutations (sequencing) - 1/6 X 10-9-9 bp bp
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Paternity testing
Mutagenesis to investigate protein function
Quantify differences in gene expression →Reverse transcription (RT)-PCR
Identify changes in expression of unknown genes→ Differential display (DD)-PCR
Forensic analysis at scene of crime
Industrial quality control
DNA sequencing
Applications of PCR
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DNA SequencingDNA Sequencing
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DNA sequencingDNA sequencing Determination of nucleotide sequence
the determination of the precise sequence of nucleotides in a sample of DNA
Two similar methods:Two similar methods:1. Maxam and Gilbert method1. Maxam and Gilbert method
2. Sanger method2. Sanger method
They depend on the production of a mixture of They depend on the production of a mixture of oligonucleotides labeled either radioactively or oligonucleotides labeled either radioactively or
fluorescein, with one common end and differing in fluorescein, with one common end and differing in length by a single nucleotide at the other endlength by a single nucleotide at the other end
This mixture of oligonucleotides is separated by This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide high resolution electrophoresis on polyacrilamide
gels and the position of the bands determinedgels and the position of the bands determined
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The Maxam-Gilbert The Maxam-Gilbert TechniqueTechnique
Principle: Principle: Chemical Degradation of Chemical Degradation of PurinesPurines• Purines (A, G) damaged by Purines (A, G) damaged by
dimethylsulfatedimethylsulfate• Methylation of baseMethylation of base• Heat releases baseHeat releases base• Alkali cleaves GAlkali cleaves G• Dilute acid cleave A>GDilute acid cleave A>G
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Maxam-Gilbert Maxam-Gilbert TechniqueTechnique
•Pyrimidines (C, Pyrimidines (C, T) are damaged T) are damaged by hydrazineby hydrazine
•Piperidine Piperidine cleaves the cleaves the backbonebackbone
•2 M NaCl inhibits 2 M NaCl inhibits the reaction with the reaction with TT
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Maxam and Gilbert MethodMaxam and Gilbert Method Chemical degradation of purified fragments (chemical degradation)Chemical degradation of purified fragments (chemical degradation) The single stranded DNA fragment to be sequenced is end-labeled The single stranded DNA fragment to be sequenced is end-labeled
by treatment with alkaline phosphatase to remove the 5’phosphateby treatment with alkaline phosphatase to remove the 5’phosphate It is then followed by reaction with P-labeled ATP in the presence of It is then followed by reaction with P-labeled ATP in the presence of
polynucleotide kinase, which attaches P labeled to the 5’terminalpolynucleotide kinase, which attaches P labeled to the 5’terminal The labeled DNA fragment is then divided into four aliquots, each of The labeled DNA fragment is then divided into four aliquots, each of
which is treated with a reagent which modifies a specific basewhich is treated with a reagent which modifies a specific base1. Aliquot A + dimethyl sulphate, which methylates guanine residue1. Aliquot A + dimethyl sulphate, which methylates guanine residue2. Aliquot B + formic acid, which modifies adenine and guanine residues2. Aliquot B + formic acid, which modifies adenine and guanine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosinecytosine The four are incubated with piperidine which cleaves the sugar The four are incubated with piperidine which cleaves the sugar
phosphate backbone of DNA next to the residue that has been phosphate backbone of DNA next to the residue that has been modifiedmodified
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Maxam-Gilbert Maxam-Gilbert sequencing - modificationssequencing - modifications
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Maxam-Gilbert sequencing: Summary
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Advantages/disadvantagesMaxam-Gilbert sequencing
Requires lots of purified DNA, and many intermediate purification steps Relatively short readings
Automation not available (sequencers) Remaining use for ‘footprinting’ (partial protection
against DNA modification when proteins bind to specific regions, and that produce ‘holes’ in the
sequence ladder)
In contrast, the Sanger sequencing methodology requires little if any DNA
purification, no restriction digests, and no labeling of the DNA sequencing template
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SangerSanger Fred Sanger, 1958Fred Sanger, 1958
• Was originally a Was originally a protein chemistprotein chemist
• Made his first mark Made his first mark in sequencing in sequencing proteinsproteins
• Made his second Made his second mark in sequencing mark in sequencing RNARNA
1980 dideoxy 1980 dideoxy sequencingsequencing
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Original Sanger MethodOriginal Sanger Method Random incorporation of a dideoxynucleoside Random incorporation of a dideoxynucleoside
triphosphate into a growing strand of DNAtriphosphate into a growing strand of DNA Requires DNA polymerase IRequires DNA polymerase I
Requires a cloning vector with initial primer Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by (M13, high yield bacteriophage, modified by
adding: beta-galactosidase screening, adding: beta-galactosidase screening, polylinker)polylinker)
Uses Uses 3232P-deoxynucleoside triphosphatesP-deoxynucleoside triphosphates
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Sanger MethodSanger Method
in-vitro DNA synthesis using ‘terminators’, use of in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain dideoxi- nucleotides that do not permit chain
elongation after their integration elongation after their integration DNA synthesis using deoxy- and DNA synthesis using deoxy- and
dideoxynucleotides that results in termination of dideoxynucleotides that results in termination of synthesis at specific nucleotidessynthesis at specific nucleotides
Requires a primer, DNA polymerase, a template, a Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection systemmixture of nucleotides, and detection system
Incorporation of di-deoxynucleotides into growing Incorporation of di-deoxynucleotides into growing strand terminates synthesisstrand terminates synthesis
Synthesized strand sizes are determined for each Synthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary di-deoxynucleotide by using gel or capillary
electrophoresiselectrophoresis Enzymatic methodsEnzymatic methods
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DideoxynucleotideDideoxynucleotide
no hydroxyl group at 3’ endprevents strand extension
CH2O
OPPP5’
3’
BASE
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The principlesThe principles Partial copies of DNA fragments made
with DNA polymerase Collection of DNA fragments that
terminate with A,C,G or T using ddNTP Separate by gel electrophoresis
Read DNA sequence
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CCGTAC3’ 5’5’ 3’primer
dNTP
ddATP
GGCA
ddTTP
GGCAT
ddCTP
GGC G
ddGTP
GGGGCATG
A T C G
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Chain Terminator BasicsChain Terminator Basics
TargetTemplate-Primer
ExtendddA
ddG
ddC
ddTLabeled Terminators
ddA
AddC
ACddG
ACG ddT
TGCA
dN : ddN100 : 1
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ElectrophoresisElectrophoresis
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Sanger Method Sequencing GelSanger Method Sequencing Gel
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Sequencing of DNA by the Sanger method
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ComparisonComparison
Sanger MethodSanger Method• EnzymaticEnzymatic• Requires DNA Requires DNA
synthesissynthesis• Termination of Termination of
chain elongationchain elongation
Maxam Gilbert Maxam Gilbert MethodMethod• ChemicalChemical• Requires DNARequires DNA• Requires long Requires long
stretches of DNAstretches of DNA• Breaks DNA at Breaks DNA at
different nucleotidesdifferent nucleotides