sba3503 lecture dna manipulation

8/2/2019 SBA3503 Lecture DNA Manipulation

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SBA3503/AQU3601

BIOTEKNOLOGIAKUAKULTUR

DNA Manipulation


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Purify

Cutting

JoiningAmplify

Probe

MarkerCloning

Expression

LibraryAssemble

Sequencing

Microarray

DNA


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DNA PURIFICATION

DNA purification can bedone using:

Gel extraction

Column purification

Chemical such asproteinase K, ethanol,RNase, DNase


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The purpose of DNA purification is toremove any contamination such asprotein, salt, excess reagent


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CUTTING DNA

Restriction endonucleases (RE) : the enzymes that areused to cut DNA molecules in specific places

Type I RE: Recognizes specific sequences in the DNAbut does not cut them

Type II RE: Recognizes and cut within specific targetsequences and therefore generate specific fragments

The name of RE: Identified firstly by the name of theorganism from which they are obtained, using the firstletter of the genus and the first two letters of thespecific name, together with a suffix indicating thespecific enzyme from that species:

E Escherichia (genus)Co coli (species)R RY13 (strain)

I The first RE identified in that bacteria


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The Nobel Prize in Physiology or Medicine 1978

"for the discovery of restriction enzymes and their

application to problems of molecular genetics"

Switzerland

BiozentrumderUniversittBasel,Switzerland

USAJohnsHopkinsUniversitySchool ofMedicine

Baltimore,MD, USA

USA

JohnsHopkinsUniversitySchool ofMedicineBaltimore,MD, USA


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Enzyme Recognition

site

No.

of

bases

Ends generated Original source of enzyme

EcoRI G/AATTC 6 5 sticky ends Escherichia coliRY13

BamHI G/GATCC 6 5 sticky ends Bacillus amyloliquefaciensH

BglII A/GATCT 6 5 sticky ends Bacillus globigii

PstI CTGCA/G 6 5 sticky ends Providencia stuartii

XmaI C/CCGGG 6 5 sticky ends Xanthomonas malvacearumSmaI CCC/GGG 6 Blunt ends Serratia marcescens

Sau3A /GATC 4 5 sticky ends Staphylococcus aureus

AluI AG/CT 4 Blunt ends Arthrobacter luteus

NotI GC/GGCCGC 8 5 sticky ends Nocardia otitidis-caviarum

PacI TTAAT/TAA 8 5 sticky ends Pseudomonas alcaligenes

Only one strand of the recognition site is shown, with aslash (/) showing the position of the cleavage site

Examples of Restriction endonuclease


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HaeIII

EcoR I

AluI

NotI

HindIII


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5 sticky ends


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An enzyme that recognizes slightly different sequence, butproduces the same ends is a isocaudomer.

Isoschizomers are pairs of restriction enzymes specific to thesame recognition sequence. For example, Sph I (CGTAC/G) andBbu I (CGTAC/G) are isoschizomers of each other.Isoschizomers are isolated from different strains of bacteriaand therefore may require different reaction conditions.

An enzyme that recognizes the same sequence but cuts itdifferently is a neoschizomer. Neoschizomers are a specifictype (subset) of Isoschizomers. For example, Sma I(GGG/CCC) andXma I (G/GGCCC) are neoschizomers of each

other.


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Restriction digestion:Incubate the DNA fragment, RE andbuffer at 37oC

After a restriction digestion, if a digest produces morethan one fragment, the fragments will normally needto be separated on a agarose gel and the desired

fragment excised and purified


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JOINING DNA

The joining or ligation of DNA fragments is carriedout by an enzyme known as DNA ligase

DNA ligase, an enzyme that catalyses the formationof a phosphodiester bond between two DNA chain

DNA ligase enzymes require a free hydroxyl group atthe 3 end of one DNA chain and a phosphate groupat the 5 end of the other. The formation of aphosphodiester bond between these groups requiresenergy

DNA ligases are only able to join DNA molecules thatare part of a double helix they are unable to jointwo molecules of single stranded DNA

Recommended ligation temperature: 4oC - 22oC


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DNA ligase has been used by researchers to join DNA

fragments to form recombinant DNA molecules


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DNA AMPLIFICATION

PCR is the amplification of specific DNA sequencesin vitro

PCR requires two primers one that is

complementary to each strand of DNA and a DNApolymerase

Repetitive heating and cooling cycles amplify theDNA between the two primer binding sites to yield

large quantities of replicated DNA

Involves 3 steps: denaturing, annealing, extension


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Thermal cycler


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PCR Cycle


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DNA CLONINGDNA cloning strategies are composed of 4 parts:

the generation of foreign DNA fragments,

the insertion of foreign DNA into a vector,

the transformation of the recombinant DNA molecule

into a host cell in which it can replicate and

a method of selecting or screening clones to identifythose that contain the particular recombinant we areinterested in.


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EXPRESSION

Expression vector: vector that used in expressionstudy of DNA fragment

Expression vector will often contain multiple

cloning site located between a strongtranscriptional promoter and terminator sequence

The expression vector will also contain an origin ofreplication and a selectable marker such that the

vactor may be autonomously replicated andmaintained within cells

Using expression vector, protein that codes by DNAfragment will be expressed/produced by the

bacteria cell


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Common host-vectorsystems that are used forprotein production: E. coli,yeast, insect and

mammalian cells

E. coliremains the host cellof choice for the majority ofprotein expression

experiments.

E. coli: rapid doubling time(approximately 20-30 min),inexpensive media, easily

broken for the harvesting ofthe proteins producedwithin the cell


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DNA MARKER

DNA marker has been used in many organismsfor population study, gender determination,selective breeding, disease detection

Various techniques: Random AmplifiedPolymorphic DNA (RAPD), Amplified FragmentLength Polymorphism (AFLP), Restriction

Fragment Length Polymorphism (RFLP),microsatellite

M1 M2 M3 F1 F2 F3


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M1 M2 M3 F1 F2 F3

malespecific

band (M) femalespecificband (F)

Identify the male specific band and female specific band cut thespecific bands purify clone identify the sequence design

specific primers amplify the DNA using the specific primers run gel

DNA markerfor male

DNA markerfor female

M1 M2 M3 F1 F2 F3


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Perak Perlis Pahang Pulau Pinang

DNA marker for population study


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DNA PROBE

DNA probe used to detect specific DNAfragment or specific clone

Nucleic acid probe (DNA or RNA), which willhybridize to the DNA sequence you are lookingfor in a specific clone

DNA sample (Southern Blot), RNA sample(Northern Blot) and protein sample (WesternBlot)


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Following gel electrophoresis, probes are often used todetect specific molecules from the mixture. However, probescannot be applied directly to the gel. The problem can besolved by three types of blotting methods:

Southern blotting

Southern blotting is a technique for detecting specific DNAfragments in a complex mixture

Northern blotting

Northern blotting is used for detecting RNA fragments,instead of DNA fragments. In the Southern blotting, DNAfragments are denatured with alkaline solution. In theNorthern blotting, RNA fragments are treated withformaldehyde to ensure linear conformation.

Western blotting

Western blotting is used to detect a particular protein in a

mixture. The probe used is therefore not DNA or RNA, butantibodies. The technique is also called "immunoblotting".


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M 1

500 bp

1000 bp

PCR

product(doublestranded)

Probe preparation

Amplification of PCR product

Denature the PCR product

PCR

product(singlestrand)


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M 123000 bp

Target preparation

Run genomic DNA

M 123000 bp

Cut genomic DNA with RE


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Southern blotting - method for the identification of DNA fragmentsthat are complementary to a know DNA sequence. It allows acomparison between the genome of a particular organism and thatof an available gene or gene fragment (the probe).

It can tell us whether an organism contains a particular gene, andprovide information about the organisation and restriction map ofthat gene.

In Southern blotting, chromosomal DNA is isolated from the

organism of interest, and digested to completion with a restrictionendonuclease enzyme. The restriction fragments are thensubjected to electrophoresis on an agarose gel, which separatesthe fragments on the basis of size.

DNA fragments in the gel are denatured (separated into single

strands) using an alkaline solution. The next step is to transferfragments from the gel onto nitrocellulose filter or nylonmembrane.

This can be performed by electrotransfer (electrophoresing theDNA out of the gel and onto a nitrocellulose filter), but is more

typically performed by simple capillary action.

In this system the denatured gel is placed onto sheet(s) of moist


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In this system, the denatured gel is placed onto sheet(s) of moistfilter paper and immersed in a buffer reservoir.

A nitrocellulose membrane is laid over the gel, and a number of dryfilter papers are placed on top of the membrane. By capillary

action, buffer moves up through the gel, drawn by the dry filterpaper. It carries the single-stranded DNA with it, and when the DNAreaches the nitrocellulose it binds to it and is immobilised in thesame position relative to where it had migrated in the gel.

The DNA is bound irreversibly to the filter/membrane by baking at

high temperature (nitrocellulose) or cross-linking throughexposure to UV light (nylon).


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The final step is to immerse the membrane in a solutioncontaining the probe - either a DNA (cDNA clone, genomicfragment, oligonucleotide) or RNA probe can be used. This isDNA hybridization - in other words the target DNA and the probe

DNA/RNA form a 'hybrid' because they are complementarysequences and so can bind to each other.

The probe is usually radioactively labelled with 32P or labelledwith special detection reagent such as enzyme horseradishperoxidase.

The membrane is washed to remove non-specifically boundprobe, and is then exposed to X-ray film (blue light sensitivefilm) - a process called autoradiography. At positions where theprobe is bound, emissions from the probe cause the X-ray film

to blacken. This allows the identification of the sizes and thenumber of fragments of chromosomal genes with strongsimilarity to the gene or gene fragment used as a probe.


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Southern blot(DNA sample)


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Northern blot(RNA sample)

Emissions from the probe causethe X-ray film/blue-lightsensitive film to blacken. Thisallows the identification of the

sizes and the number offragments of chromosomalgenes with strong similarity tothe gene or gene fragment usedas a probe

RNA samples


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Colony blotting


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The principle involved is that the library (in form ofbacterial colony) is replicated onto a filter, which isthen treated to release the DNA and bind it to the

filter, which then carries a pattern of DNA spotsthat replicates the position of the colonies on theoriginal plate.

The filter is then hybridized with the probe, whichhas first been labelled so that it can be easilydetected.

This allows us to detect which DNA spots hybridize

to the probe and recover the corresponding clonesfrom the original plate


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LIBRARY

A genomic library: a collection of cloneswhich between them represent the entiregenome of an organism

A cDNA library: a collection of clones thatconstructed from DNA copies of themRNA present in the originating cells at

the time of isolation


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The first step in producing a genomic library is tofragment the genomic DNA into pieces of a suitablesize for cloning in an appropriate vector.

To construct a library of overlapping fragments is touse partial digestion

This means using conditions, such as shortdigestion times, that result in only a smallproportion of the available sites being cut

A similar effect can be obtained by using very smallamounts of enzyme, or by incubating the digest atreduced temperature


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The digested material is then fractionated byelectrophoresis to obtain fragments of therequired size range before cloning in anappropriate vector

Restriction

sites GenomicDNA

Collectionofoverlappingfragments


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mRNAtemplate

First strandcDNAsynthesis

Second

strand cDNAsynthesis

Ligation withEcoRI

adapter

Digest withXhoI

XhoI

Synthesis of cDNA strand for cDNA library construction


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Each colony containsdifferent DNA fragment


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DNA ASSEMBLE

Process to assemble DNA sequences usingbioinformatics analysis in order to producea consensus

Restriction


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Restrictionsites

GenomicDNA

Collectionofoverlapping

fragments

Consensus


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CTAGACCGTAGACTAGCAGAGATCAGTACGATAGCCAGTAGCAGAATGCCAGTA

ATGCGTGACTAGACCGTAGACTAGCAGAGATCAGTACGACTAGACCGTAGACTAGCAGAGATCAGTACGATA

GTGACTAGACCGTAGACTAGCAGAGATCAGTACGATAGCCAGTAGC

ATGCGTGACTAGACCGTAGACTAGCAGAGATCAGTACGATAGCCAGTAGCAGAATGCCAGTA

Consensus

Assemble


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DNA SEQUENCING

The most fundamental way of analyzing the structureof DNA, whether it is a recombinant plasmid, anatural gene or a whole genome is to determine the

sequence of bases of which it is composed

The advent ofautomated DNA sequencing machineshas made the determination of the sequence of

individual fragments very much faster and morereliable and the dramatic increase in the availablecomputer power has enabled the assembly of verylarge numbers of fragments

ATGCGTGACTAGACCGTAGACTAGCAGAGATATTGGCTGACATACA

DNA SEQUENCING


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ABI Prism GeneticAnalyser

ELECTROPHORESIS:

Slab gel Capillary system

DNA SEQUENCING


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C

G

A

T

Capillary electrophoresis:

Small amount of sampleDetection system is more sensitive

Faster

Result more consistent


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1963

F. Sanger of Britain developedsequencing procedure for

proteins

2000

J. Craig Ventor, along withFrancis Collins, jointly announce

the sequencing of the entirehuman genome


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Sample preparation - (PCR product / plasmid)

Cycle sequencing

Sample purification

Automated sequencing

Sequence analysis

DNA SEQUENCING

S l ti


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Sample preparationIn 0.2 ml tube, add:

3.0 ul sample (DNA plasmid)8.0 ul ABI BigDye Terminator ready reaction mix

1.0 ul primer (5 pmol)

8.0 ul deionized water

Total volume: 20 ul

Cycle sequencing:

96o

C - 5 min96 oC - 30 s

50 oC - 15 s

60 oC - 4 min

10 oC -

50 cycles

C


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G

A

T

Cycle sequencing


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Thermal cycler Sequencer


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Cycle sequencingusing thermal

cycler

ddNTP (dideoxynucleotide triphosphate:

ddATP,ddGTP,ddCTP,ddTTP)

BigDye terminator

S l ifi ti


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Sample purificationCycle sequencing product, add:

50 ul ethanol (95% ETOH), 2 ul 3M sodium acetate(pH 4.6)

Vortex, incubate at room temperature for 15 min

Centrifuge at 14,000 rpm for 30 min at 4 oC

Remove supernatant

Add 250 ul ethanol (70% ETOH) to the pellet

Vortex, Centrifuge at 14,000 rpm for 5 min at 4 oC

Remove supernatant

Add 250 ul ethanol (70% ETOH) to the pellet

Vortex, Centrifuge at 14,000 rpm for 5 min at 4 oC

Remove supernatant

Dry the pellet


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Purified pellet, add:

10 ul formamide HIDI (Applied Biosystem, USA) todissolve the pellet

Denature the DNA strand at 95 oC for 7 min

Short spin the tube

Samples are ready to analyze in sequencer



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ABI PRISM 3100 Genetic Analyzer

SEQUENCER

Sample A


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pSample B

Detector in sequencer machine will detect thefluorescence dyes based on the wave length of each

fluorescence dye and these data will be transformedinto a graph (chromatogram) by computer software

Sample A

Sample B



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Automated sequencing using sequencer machine (eg.ABI PRISM 3100 Genetic Analyzer)

Automated sequencing involves electrophoresis ofsamples from cathode to anode through a specialmedium called polymer (eg. POP6, Applied

Biosystems, USA)

The migration of sample will be detected by asensitive detector that connects to a CCD camera that

detects the fluorescence dye. The data will betransformed into a chromatogram file by computersoftware.

The chromatogram file can be used to characterizethe sequence using bioinformatics analysis


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Unpurified samplePurified sample


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The Human Genome Project (HGP) is an international13-year effort formally begun in October 1990. Theproject was planned to last 15 years, but rapidtechnological advances accelerated the completion to2003.

Project goals were to determine the complete sequenceof the 3 billion DNA subunits (bases), identify all humangenes, and make them accessible for further biologicalstudy. As part of the HGP, parallel sequencing was done

for selected model organisms such as the bacterium E.colito help develop the technology and interpret humangene function. The Department of Energy's HumanGenome Program and the National Institutes of Health'sNational Human Genome Research Institute (NHGRI)together sponsored the U.S. Human Genome Project.

HUMAN GENOME PROJECT



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Approximately, 10 - 20 human DNA samples used inHuman Genome Project and the identity of thesamples are still remain unknown

Th bj ti f H G P j t


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The objectives of Human Genome Project:

To determine all the 3 billions (3 x 109) base in thehuman genome

To determine all the 30,000 genes in human genome

To determine the function of all the 30,000 genes

To store all the data into one appropriate database

To catalyze the development of new biotechnologytechnique and new medicine


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DNA MICROARRAY

Large scale


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g

Multi channel micropipette

96 well Microplate

DNA library

Large scale plasmid extraction


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Glass slide that contains microscopic elements thatallow it binding to DNA, gene or gene product

DNA microarray is a very effective method indetermining expressed genes in one experiment

Biochips, DNA chip or gene chip/microarraycontains a collection of small elements in row andcolumn

DNA MICROARRAY


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The Affymetrix GeneChip Zebrafish GenomeArray can be used to study gene expression ofover 14,900 Danio rerio transcripts.

Human, Mouse, Yeast

Commercial Genechipin the market:

Affymetrix GeneChip


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DNA spotting onto glass slideusing robotic machine


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Data analysis based on the

intensity of fluorescencedye of DNA spot

Cy5 Cy3

Scanner


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Image scan at 650 nm Cy5(0 hr)

Image scan at 550 nm Cy3(16 hrs)

Overlay image

Red spot:Gene

expressed in

sample: 0 hr

Yellow spot:

Genes expressedin both samples

Green spot: Geneexpressed in

sample: 16 hrs

Experiment: Determination ofexpressed gene in fishes that have

been cultured at 10o

C for 16 hrs


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Data analysis based on the color

intensity of DNA spot


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sba3503 lecture dna manipulation

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