CLONING SIMULATION PROJECT 2003

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CLONING SIMULATION PROJECT 2003

• CLONING OF HUMAN VASCULAR ENDOTHELIAL GROWTH
  FACTOR (VEGF)
• GROUP H

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SUPERVISOR PROF MADYA DR ZAINUL F.
ZAINUDDINGROUP H

• Liew Chyi 72748
• Norayuni Mohamed 70957
• Norazlin Abdul Aziz 70958
• Phong Boon Kien 70962
• Sharifah Nur Hafiza Syed A.Rahman 71060
• Siti Balkiah Ismail 71061
• Siti Hasmah Ab. Rahim 71062

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OBJECTIVES

• To clone a gene of interest with potential values
• To undergo all process of cloning a gene in
  molecular biology by means of bioinformatic tools
• To apply bioinformatic tools in molecular biology
  work field

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THE GENE OF INTERESTHUMAN VASCULAR ENDOTHELIAL
GROWTH FACTOR (VEGF)

• Briefly about the gene..
• A component of growth hormone
• Promotes angiogenesis (formation of new blood
  vessels)
• Is a mitogen for vascular endothelial cells
• Mitogen any substance that can cause cells to
  begin division

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WHY DO WE PICK THIS GENE?

• This gene can promote as well as inhibit
  angiogenesis
• Help in treating blocked blood vessel diseases,
  coronary heart disease
• Anti-cancer therapeutics
• High commercial value demanding market

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PROCEDURE OF CLONING SIMULATION PROJECT
1.Hunt for gene
5.Compare gene sequence
2.Getting gene sequence
6.Design primer probe
3.Look for ORF
7.Cloning strategy
4.Restriction analysis
8.Screening method
9.Protein purification
10.Proteins commercial value
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STEPS IN CLONING THE GENE OF INTEREST
1.Choosing a suitable vector pETBlue-2
2.Preparation of cDNA library
3.PCR amplification Amplification of VEGF
cDNA
4.Restriction enzyme digestion PCR product
and pETBlue-2 treated with enzymes EcoR1 and Xho1
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5.Ligation of PCR products into pETBlue-2 vector
Enzyme Ligase
6.Transformation Host cells E.coli
(BL21) Calcium chloride method
7.Verification of successful cloning
8.Protein expression
9.Protein purification
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THE GENEVEGF

• VEGF Vascular Endothelial Growth Factor
• Source Homo sapiens
• Consisting 215aa
• 648bp
• Coding sequence 1648bp
• mRNA, linear

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ORF ANALYSIS

• ORF A DNA sequence that contains series of
  codon that can be translated into functional
  protein
• Criteria for ORF Must consist start codon (ATG)
  and stop codon (TGA,TAG or TAA)

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ORF
ORF chosen is coloured in pink
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THE AMINO ACID SEQUENCES
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INTERPRETATION OF ORF ANALYSIS

• From the ORF analysis, we have chosen the longest
  frame
• Frame consist of 648bp
• 1 frame (forward strand)
• contain start and stop codon
• Accession NP_003367
• NM_003376

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MOLECULAR WEIGHT

• Molecular weight of (648/3) x 110
• VEGF protein 23,760 Daltons
  Approximately 23,000 Daltons
• Approximately 23 kDa

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RESTRICTION ENZYME ANALYSIS

• To determine enzymes which DO NOT CUT within the
  gene sequence
• Important because avoid the gene sequence from
  being cut
• The whole sequence is needed for the cloning
• The enzyme should only cut at LINKER and vector
  of cloning

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These nucleases do not cut the gene sequence

• AatI, AatII, Acc113I, Acc16I,
  Acc65I, AccB1I, AccB7I, AccI,
  AccIII, AclNI, AcsI, AcyI, AfeI,
  AflII, AgeI, AhdI, Alw21I,
  Alw44I, Ama87I, AocI, Aor51HI,
  ApaI, ApaLI, ApoI, AscI, AseI,
  AsnI, Asp700I, Asp718I, AspEI,
  AspHI, AspI, AtsI, AvaI, AviII,
  AvrII, BalI, BamHI, BanI,
  BanII, BanIII, BbeI, BbiII,
  BbrPI, BbsI, BbuI, Bbv12I,
  Bbv16II, BcgI, BclI, BcoI, BfrI,
  BglI, BglII, BlnI, BlpI, BpiI,
  Bpu1102I, Bpu14I, BpuAI, Bsa29I,
  BsaAI, BsaHI, BscI, Bse118I,
  Bse21I, BseAI, BseCI, BsePI,
  BshNI, BsiHKAI, BsiMI, BsiWI,
  BsmBI, BsoBI, Bsp106I, Bsp119I,
  Bsp120I, Bsp13I, Bsp143II, Bsp1720I,
  Bsp68I, BspDI, BspEI, BspHI,
  BspLU11I, BspMI, BspTI, BspXI,
  BsrFI, BssAI, BssHII, Bst1107I,
  Bst98I, BstBI, BstEII, BstH2I,
  BstI, BstPI, BstSNI, BstXI,
  BstZI, Bsu15I, Bsu36I, CciNI,
  CelII, Cfr10I, Cfr42I, Cfr9I,
  CfrI, ClaI, CpoI, Csp45I, CspI,
  CvnI, DraI, DrdI, EaeI, EagI,
  Eam1105I, Ecl136II, EclHKI, EclXI,
  Eco105I, Eco147I, Eco24I,
  Eco255I, Eco32I, Eco47III, Eco52I,
  Eco57I, Eco64I, Eco72I, Eco81I,
  Eco88I, Eco91I, EcoICRI, EcoO65I,
  EcoRI, EcoRV, EcoT22I, EheI,
  Esp1396I, Esp3I, FauNDI, FbaI,
  FriOI, FseI, FspI, HaeII, Hin1I,
  HincII, HindII, HindIII, HpaI,
  Hsp92I, KasI, Kpn2I, KpnI,
  Ksp22I, KspI, LspI, MfeI, MluI,
  MluNI, Mph1103I, MroI, MroNI,
  MscI, Msp17I, MspCI, MunI, NaeI,
  NarI, NdeI, NgoAIV, NgoMI,
  NheI, NotI, NruI, NsiI, NspV,
  PacI, PaeI, PaeR7I, Pfl23II,
  PflMI, PinAI, PmaCI, Pme55I,
  PmeI, PmlI, Ppu10I, PpuMI,
  PshAI, PshBI, Psp124BI, Psp1406I,
  Psp5II, PspAI, PspALI, PspEI,
  PspLI, PspOMI, PstI, PstNHI,
  PvuII, RcaI, RsrII, SacI, SacII,
  SalI, SapI, SbfI, ScaI, SexAI,
  SfiI, Sfr274I, Sfr303I, SfuI,
  SgfI, SgrAI, SmaI, SmiI, SnaBI,
  SpeI, SphI, SplI, SrfI,
  Sse8387I, SseBI, SspI, SstI,
  SstII, StuI, SunI, SwaI, Tth111I,
  Van91I, Vha464I, VneI, VspI,
  XbaI, XcmI, XhoI, XmaI, XmaIII,
  XmnI, Zsp2I

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COMPARING GENE SEQUENCES

• BLAST analysis
• To confirm that the gene really codes for human
  VEGF
• To identify other genes similar to our gene of
  interest
• Result gtgi30172563refNM_003376.3 Homo
  sapiens vascular endothelial growth factor
  (VEGF), mRNA
• Length 1723
• Score 1237 bits (624), Expect 0.0
• Identities 640/648 (98)
• Strand Plus / Plus
•  

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BLAST RESULT.
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MULTIPLE DNA SEQUENCE ALIGNMENT..

• Compare DNA sequences from BLAST analysis result
• Shows identities, similarities and differences of
  the genes selected
• Organisms for comparison purposes are a chosen
  randomly
• Canis familiaris, Rattus norvegicus, Felis catus
  and Bos taurus

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Phylogenic tree
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Choosing of Vector

• Autonomously replicating extrachromosomal
  circular genetic material
• pETBlue-2

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Advantages

• Function as cloning and protein expression vector
• Compatible with the protein expression mechanism
  in E.coli host cells
• Ampicillin resistance marker
• Blue/white visual screening
• Expression is made possible by T7 transcription
  and translation signals
• High copy number
• No basal expression
• Available as blunt ends and sticky ends gene
  insertion
• C-terminal HSV Tag epitope and His Tag sequence
  for protein purification

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• For recombinant cloning propose, cDNA was used
• By transcript the mRNA using reverse
  transcriptase, the amplified by PCR
• Directly obtained from cDNA library and amplified
  by PCR method

RT-PCR

• Conventional RT-PCR consists two steps
• Transcript the mRNA into cDNA

• First strand cDNA synthesis

• oligo (dT) primer
• Reverse transcriptase, which traditionly have
  been isolated from viruses
• Common commercially available reverse
  transcriptase is Moloney Murine Leukimia Virus
  (MMLU)
• Initiating polymerization by adding
  nucleotides to the nacent polynucleotide oligo
  (dT) primer in the 5 to 3 direction using mRNA
  as the template
• Product mRNA-DNA hybrid

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RT-PCRcont

• Second strand cDNA synthesis

• Introduce nicks into the RNA half of this
  DNA.RNA hybrid
• By RNAse H, which exhibits endonucleolytic
  cleavage of the RNA moiety of mRNA-DNA hybrids,
  as well as 5 to 3 and 3 to 5 exoribonuclease
  activity
• With adding of DNA polymerase, these RNA
  fragments serve as primers for the second
  strand cDNA synthesis
• Okazaki fragments ligate by T4 DNA ligase
• Product double-stranded cDNA

• Amplified cDNA by PCR

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Polymerase Chain Reaction (PCR)

• In principle, generate any amount of DNA from a
  single molecule specifically and highly
  accurately amplify a particular DNA sequence
  without needing a clone vector

3 basic steps in perform PCR

• Heat the DNA template to denature
• Hybridize or anneal primers that flank area of
  interest
• DNA polymerase binds and extends primer

This series of 3 steps is called one cycle, and
is repeated 20-30 time
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Factors Influencing the PCR Products

• Mg2 Concentration

• dNTP concentration of about 200µm each are
  usually recommended for the Taq polymerase, at
  1.5 mM MgCl2
• Besides the Mg2 bound by the dNTP and the
  DNA, Taq polymerase requires free magnesium to
  work properly

• Template DNA Concentration

• Too much template may increase the amount of
  contaminants and reduce efficiency

• Enzymes used-Taq DNA polymerase

• Taq DNA polymerase, a thermostable DNA
  polymerase purified from the thermophilic
  bacterium, Thermus aquaticus, that can survive
  extended incubation at 95ºC
• Catalyzes the incorporation of dNTPs into
  DNA, therefore extend the gene specific primer
• 5 to 3 exonuclease activity, but lack of 3
  to 5 exonuclease activity

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• Primer

• Length of 18-24 bases are enough specific
• Annealing temperature of at least 50ºC
• Absolutely no intra or inter-primer homology
• 45 and 55 GC content
• Forward primer should not be complementary to
  reverse primer

Primer Design
cDNA 5atgaactttctgctgtcttggtgacaagccgaggcgg
tga 3 Restriction enzyme cutting
site EcoRI 5G/AATTC 3 XhoI 5C/TCGAG
3 Forward primer 5 AGCGAATTC
ATGAACTTTCTGCT 3 Reverse primer 5
TATCTCGAG TCACCGCCTCGGCT 3 Melting temperature,
Tm (AT)2(GC)4 Annealing temperature Tm-2?C

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Steps of PCR
1. DNA denaturation
2. Primer annealing
 3.Extension (end)        
3. Extension (beginning)
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Restriction Enzyme Digestion

• After the PCR amplification, our products i.e.
  the gene of interest, VEGF, contains EcoRI
  restriction enzyme recognition sequence at the 5
  ends and XhoI recognition sequence at the 3
  ends.
• To clone the PCR products into the plasmid
  selected, pETBlue-2, both PCR products and the
  plasmids need to be treated with EcoRI and XhoI.
• The action of these enzymes is to recognize the
  specific cutting site and cut at the DNA sequence.

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• This restriction enzyme digestion will produce
  stricky ends at the both ends of our PCR products
  and also at the multiple cloning site of
  pETBlue-2.
• EcoR1 recognition sequence
• G/AATT C
• C TTAA/G
• XhoI recognition sequence
• C/TCGA G
• G AGAT/C

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LIGATION

• The ligase enzymes catalyze the formation of a
  phosphodiester bond between adjacent 3-OH and
  5-P termini in DNA.
• Sticky end can only join with compatible sticky
  ends whereas blunt ends can join with any blunt
  ends but not with sticky ends.
• Ligation of complementary sticky ends is much
  more efficient compare to the ligation of two
  blunt ends.

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• ligation of complementary overhangs of sticky
  ends allows hydrogen bonds to form and stabilize
  the two molecules before ligase can act.
• We use T4 DNA ligase for the ligation purpose in
  this project.
• T4 DNA ligase also ligate fragments with blunt
  ends, however higher concentration of the enzyme
  are usually recommended for this purpose.
• Ligation buffer contains ATP is used because T4
  DNA ligase need ATP supplement to work
  efficiently.

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MATERIALS AND PROTOCOL

• In a ligation reaction , the vector and insert
  ration should be in the ratio 13.
• We use the EcoRI and XhoI to cut pETBlue-2 and
  VEGF insert DNA for the ligation experiment.

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METHODOLOGY

• To a sterile microfuge place the following in
  order
• 10x Ligase buffer 2µl
• Sterile deionised water 10µl
• EcoRI cut pHis plasmid 1µl
• VEGF insert (EcoRI and 3µl
• XhoI restricted )
• PEG 4000 (polyethylene 2µl
• glycol 4000)
• T4 DNA Ligase (2 units/µl) 2µl
• Total 20µl

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• Incubate at room temperature for 1 hour (
  Incubation at 16oC overnight also can be done but
  will not be used for this cloning project)
• After incubation, store the ligation mix at
  -20oC. It will be used for transformation.

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TRANSFORMATION

• The transformation experiment done is based on
  the observations of Mandel and Higa (1970), Cohen
  et al., (1972), and Oishi and Cosloy (1972).
• Transformation is the process of uptake of DNA
  without cells to cells contact.
• Most species of bacterial, under normal
  circumstance very rarely takes up foreign DNA.
• For the bacteria that are not natural competence
  for transformation, some artificial methods need.

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• Physically or chemically treatment can be done.
• There are two method provided, Calcium Chloride
  method and electroporation.

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• The Calcium Chloride method was first introduced
  by Mandel and Higa (1970).
• Bacteria treated with ice-cold solution of
  Calcium Chloride and then briefly heated could be
  transfected with bacteriophage DNA.
• Electroporation method use an electroporator to
  produce small holes in the membranes of cells
  allowing DNA to enter.
• The method of electroporation was developed
  originally to introduce DNA into eukaryotic
  cells.
• Recently been used to transform E. coli and other
  bacteria.

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• The transformation efficiencies is about 109 1010
  transformants / µg of DNA.
• Better transformation efficiency can be achieved
  by optimizing various parameters.
• These parameters including the strength of the
  electrical field, the length of the electrical
  pulse, and the concentration of DNA.
• The higher levels of transformation were achieved
  with a combination of higher voltages or longer
  pulses.
• It will decrease the cell viability, that is may
  resulted in 50 75 cells death.

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• We used Calcium Chloride method in this cloning
  project.
• The objective is to transform the recombinant
  plasmid, which contains VEGF gene insert, onto an
  E. coli host BL21(DE3).

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MATERIALS AND PROTOCOL

• 3 tubes of competent cells are prepared (always
  kept on ice).
• Each tube contains 150?l of E.coli competent
  cells.
• The tubes are labeled as T (test), V (vector) and
  C (control).

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SCREENING METHOD
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BLUE WHITE CLONING METHOD

• Ampicillin resistant marker
• E.coli contain vector grow
• E.coli without vector dont grow
• white colonies contain inserts
• blue colonies contain religated vector

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pETBlue-2
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Examples of light blue (LB), white (W) and blue
(B) colonies.
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PCR SCREENING METHOD

• To make sure that we had cloned the
• right gene.
• Sample tube shows a band on the gel exactly
  same as the previous one.
• Negative control no band
• free from contamination.
• Positive control
• function as indicator to show that our
• agarose gel is well prepared.

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Restriction Enzyme Digestion

• Use the same enzymes EcoR1 and Xho1
• Run in agarose gel
• Insertion determined by comparison with the
  migration of DNA fragment of known size
• See the molecular weight of gene and plasmid

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DNA SEQUENCING

• We use the Sanger dideoxynucleotide chain
  termination method.
• Need several composition
• 1. ddNTP ddATP, ddCTP, ddGTP, ddDTP
• 2. DNA polymerase
• 3. one primer
• 4. template DNA
• 5. labeling

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HOW TO DO ???????

• Put all the composition in four tubes with
  different ddNTP in each tube.
• Resolved by electrophoresis and read the sequence
  from the bottom of the gel.

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• ddNTPs lack both the 2 and 3 hydroxyl group.
• 3 hydroxyl group required for the formation of
  phosphodiester bond.
• The presence of ddNTPs cause chain termination.
• Produce all possible size fragments

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PROTEIN EXPRESSION
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PROTEIN PURIFICATION
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Purification of the protein (Nickel Affinity
Chromatography Method)

• 1.Before purifying the Vascular Endothelial
  Growth Factor(VEGF),lysis the cells to release
  the mixture of the protein.
• 2.Vascular Endothelial Growth Factor(VEGF)
• attach with 6 His.tag
• 3.Pass the mixture of the protein through a
  Nickel affinity chromatography column (His.bind
  column 10mg/run)
• 4.Wash the protein without the His.tag
• or protein that we dont want with buffer.

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• 5.Elute the VEGF with His.tag from column by a
  lower pH buffer.
• 6.Treat His.tag protein with the specific
  protease to cleave off the His.tag from VEGF.
• 7.Running the VEGF over the His.tag column again
  for freed of the His.tag peptide from VEGF.

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Nickel affinity
His tag
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USE OF PROTEIN1. As a principal regulator of
blood vessel formation and haematopoiesis2.
Have a regulatory effects on certain blood
cells.It may promote monocyte chemotaxis3.
Surgical procedures4. Therapy for the treatment
of peripheral vascular disease
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COMMERCIAL VALUEVEGF are being developed to
treat diseases of blocked blood vessels,
including coronary heart disease, while the
latter are promising primarily as anti cancer
therapeutics.
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CONCLUSION..

• The VEGF gene is a very high potential gene
  medical field
• We hope , in future there will be further
  research conducted to determine and establish
  treatments for various diseases (esp. cancer)
  using this gene.
• This project has given a better understanding and
  view of the importance of molecular biology field
  in our daily lives.

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ACKNOWLEDGEMENTS

• We would like to thank
• Our supervisor , Prof Madya Dr Zainul F.
  Zainuddin for his cooperation in this project.
• Our lecturers , Dr. M. Ravichandran, Dr. Shaharum
  and others for their help in accomplishing our
  project.
• To others who helped us in any kind of way.

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THANK YOU