recombinant DNA Technology.ppt - mums.ac.ir

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History of recombinant DNA technology

Recombinant DNA Technology ((DNA N cloning) g)

Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists i i namedd Boyer andd Cohen h in i 1973.

Majid Mojarrad

Recombinant DNA „

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Fig. 1.2 Many scientific disciplines contribute to molecular biotechnology, which generates a wide range of commercial products

Formed when two genes from two different sources often different species are combined in vitro into the same molecule This pprocess is called ggenetic engineering g g Lead to a new field called biotechnology, the manipulation of organisms or their components to make useful products

DNA cloning gene cloning - prepare multiple identical copies of gene-sized pieces of DNA.

Generation of DNA fragment

Joining into a vector or carrier molecule

Introduction into host cell to amplification

Selection of required sequences

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Genes can be cloned into recombinant DNA vectors „

Five steps: ‰ ‰ ‰ ‰ ‰

Isolation of vector and gene Insert gene into vector Transform bacteria with vector Plate bacteria under selection Identify clones carrying vector

Generation of DNA fragment

Joining into a vector

Introduction into host cell

Mechanical shearing

Blunt end ligation

Transformation with recombinant plasmid

Restriction enzymatic digestion

Sticky end ligation

Direct chemical synthesis

Homopolymer tailing

PCR

Use of linker

RT-PCR

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Enzymatic digestion of purified DNA PCR RT-PCR

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Transformation Electroporation Conjugation

symmetrical series of four to eight bases Sticky or blunt ends Restriction fragments-specific for each DNA

Origin – bacteria ‰

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in vitro

Cut DNA at specific DNA sequences

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Methods of Introducing Foreign DNA

Packaging DNA

Restriction enzymes are used to make recombinant DNA

Generation of DNA fragment „

Ligase independent cloning

Transfection with recombinant phage

How do they protect their own DNA?

Combined by DNA ligase

How can you identify which clones carry the vector? 1) Nucleic acid hybridization 2) Isolation of plasmid DNA from individual clones and restriction mapping PCR

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PCR: History PCR Invention: 1987 Kary Mullis PCR is essentially DNA replication in a tube.

Series of repetitive steps enabling amplification of target DNA from a complex mixture of DNA

The major advantages of PCR „

Speed and ease of use „

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30 cycles each taking 3-5 mins

Sensitivity „

Need for Target DNA sequence information „

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Can amplify from a single cell, cell great care must be taken to avoid contamination

There is an upper pp limit to the size of DNA synthesized y byy PCR

Infidelity of replication „

Will even work on degraded DNA or fixed DNA

To construct primers you need to know your target

Short size limit for product „

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Robustness „

Disadvantages of PCR

Because the PCR polymerases are heat stable they ten not to have the 3’->5’ exonuclease activity

Basics „

Target

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dNTP’s

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Buffer

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Primers „

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DNA Taq polymerase

Denature-- 920C-950C Denature (940C) Anneal- 500C-720C AnnealAim for 50C below calculated Tm (520C-580C generally best) Extension - 680C-800C (720C) highest efficiency 700C-800C

Template Plasmid „ cDNA (RT (RT--PCR) „ Genomic DNA „

P

C

Plasmid

P

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Purified (P) Crude Lysate (C)

C

Genomic

40ng 10ng 1ng

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dNTPs

Buffer All 10x 10x Buffers are not the same

Mixture of dATP dATP,, dCTP, dCTP, dGTP, dGTP, dTTP or dUTP Purity-- chemical or enzymatic synthesis Purity Stability-- concentration – Li or Na salt form Stability

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Salt

1010-50 mM Tris pH 8.3

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Monovalent cation

100100-150 mM KCl or NaCl

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Divalent cation Mg22+, Mn2 Mg Mn2+

1.5uM or > MgCl2+

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Buffer Systems „

Modifications: Mg pH Ionic strength Additives

Detergent, Glycerol, Gelatin

Buffer Additives „ „ „ „

Mg2+

Additives

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Q-solution solution--Betaine DMSO BSA Glycerol Gelatin PEG GC--melt GC Formamide Detergents

Q

D

B

G

P Q/D F

D

Ionic strength

Primers

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Additional Considerations

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Pair complementary to opposite strands 5’ 3’ sense primer 3’ 5’ anti anti--sense primer

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Secondary structurestructure- avoid hairpins, self self--dimers dimers,, cross cross-homology

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Features 18--26 nucleotides 18

Equal mix GC to AT bases

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Avoid di di--nucleotide repeats that occur consecutively consecutively-ATATATAT

Match Tm of primers

Tm oC= 2(A/T) + 4(G/C)

3’ Stability

GG or GC clamps

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Avoid long runs of single basesbases- ACGGGGGGAT

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Avoid crosscross-homologyhomology- BLAST Test

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Primer Variation Example PCR 1st Round vary primer pairs Sets A-F A= Primer 1F B= Primer 2F C= Primer 3F D= Primer 1F D E= Primer 2F F= Primer 3F Forward primers Primer 1: GAGGGCAGATTCGGGAATG Primer 2: TCGGGAGAGGCCCTTCCC Primer 3: CAGTTTCCCGGGTTCGGC

Primer 1R Primer 1R Primer 1R Primer 2R Primer 2R Primer 2R

DNA Taq Polymerases Considerations: Aim of experiment „ „ „

Thermal stability Processivity Fidelity

Tm=600c Tm=620c Tm=600c

Reverse primers Primer 1: AGCCTAATCAAGTCACTATCAAG Primer 2: GCAAGTGAGAAAATGGGGAG

Tm=620C Tm=600C

DNA Taq Polymerases Standard polymerase Standard polymerase with loading dye Hot Start polymerase

works for most applications

Polymerase blends or cocktails

combine polymerases for fidelity with speed

aids in higher throughthrough-put inhibits non non--specific primer extension

Fidelity „PCR

product sequence

„PCR product T/A cloned Individual isolates sequenced

Taq blend

Standard Taq

Hot Start Taq

PCR Cycling

Modified PCR Methods „

Hot Start PCR Manual Hot Start Physical Barrier M difi d Taq Modified T DNA polymerase l Oligo Inhibitors Modified dNTP’s

Semi-Nested or Nested PCR Semi„ Touch down PCR „

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Semi--Nested or Nested Semi Nested--PCR

Additional PCR Methods „

„Specificity

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„Sensitivity

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Allele-specific PCR AlleleAssembly PCR (PCA) Breakpoint PCR Intersequence--specific PCR (ISSR) Intersequence Inverse--PCR (IPCR or REInverse RE-PCR) Ligation Mediated PCR (LM(LM-PCR) Long distance PCR Multiplex--PCR Multiplex Methylation Specific PCR Mini--primer PCR Mini Quantitative PCR or RealReal-time PCR Reverse Transcriptase PCR (RT(RT-PCR)

RT--PCR RT „Quality

Multiplex--PCR Multiplex

of RNA

„Increase „Increase

„Reverse Transcriptase-QC

oligo dT random hexamers gene specific primers

throughput data with limited material 1

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3

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5

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7 8

Exon 7 and 8 Exon 9 Exon 3 Exon 5 Exon 1 Exon 2 Exon 6 Exon 4

Long--PCR Long „Analyze „Tool

large area in single reaction

to analyze inserts and breakpoints 14kb 3kb

20kb

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1.6kb

Breakpoint--PCR Breakpoint „

Isolate low frequency event

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Inverse--PCR and REInverse RE-Inverse PCR „

Restriction fragment length polymorphism

Isolate unknown flanking region Digest with restriction enzyme Ligate with T4 T4 DNA ligase

Allele specific PCR

TaqMan™ TaqMan™ assay

Genomic Walking

Real--Time PCR or Q Real Q--PCR „ „

Allele specific PCR using the 5’ to 3’ exo activity and a third primer with a Fluor and dQ Quencher. h

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Increased Sensitivity Increased Specificity Increased Throughput

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Designing of primers „

Identification of restriction map:

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Primer Design

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Evaluation of primers specifity

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PCR reaction

http://www.firstmarket.com/cutter/cut2.html http://frodo wi mit edu/primer3/ http://frodo.wi.mit.edu/primer3/ http://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome

Introducing of restriction sites

Cloning of PCR products

PCR cycle

transformation Plasmid digestion „ PCR product digestion „ Measurement of molar concentration of DNA htt // http://www.basic.northwestern.edu/biotools/olig b i th t d /bi t l / li ocalc.html „ Ligation „ transformation „

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Heat shock electroporation

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Preparing medium Antibiotic selection Liquid culture Pl Plasmid id extraction t ti Plasmid identity confirmation Sequencing subcloning

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Extraction of plasmid