GENE CLONING: overview

Transcription

1 TMMO504: Laboratory Molecular Tropical Medicine and Genetics GENE CLONING: overview Instructor: Asst.Prof.Dr. Santi Maneewatchararangsri Department of Molecular Tropical Medicine and Genetics, Mahidol University

2 Resources Available from: [Accessed 1 May 2018]

3 Citation: Maneewatchararangsri S. GENE CLONING: overview. [Lecture] TMMO504: Laboratory Molecular Tropical Medicine and Genetics,. 19 April April 2018 Available from: Accessed 19 April 2018.

4 Objectives Describe general idea of molecular cloning: Target DNA, Vector, and Host Describe the following steps for molecular cloning: 1. Identification& selection of target DNA sequence/gene 2. Preparation of target DNA sequence/gene (coding) 3. Cloning of target DNA sequence to storage system (Next)

5 Objectives Describe the following steps for molecular cloning: 4. Selection of positive DNA sequence 5. Subcloning of the target DNA to expression system 6. Selection of positive DNA clone 7. Characterization/ applications

6 MOLECULAR CLONING = DNA sequence + vector (which can replicate in host) and this host can produce multiple copy of inserted DNA compatibility of DNA, vector and host

7 DNA SEQUENCE PCR product : Gene specific genomic DNA : Library construction complementary DNA (cdna) : synthesize from mrna

8 VECTOR in cloning = DNA molecule carry the DNA SEQUENCE of interest into the host cell & facilitates its replication

9 TYPE OF VECTOR 1. Plasmid for small segments of DNA (10-15 kb) P lac 5 MCS 3 MCS GFP pgfpuv 3.3 kb circular dsdna can insert or more DNA segments (10-15 kb) autonomous propagate within host cell contain origin of replication (ORI): a region of DNA that allows multiplication of the plasmid

10 puc18/19 Plasmid ORI Multiple cloning site (MCS) Drug resistant marker

11 pet23a Plasmid: circular map

12 pet23a Plasmid: Linear map

13 Pcantab5e phagemid: Linear map

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15 2. Cosmid contain propagated plasmids features containing cos sequences from bacteriophage λ ORI & an antibiotic resistance marker can insert up to 45 kb of foreign DNA can be propagated as plasmid

16 3. Bacteriophage λ viruses that infect bacteria dsdna sequence of ~ 50 kb can propagate in vitro can insert large pieces of foreign DNA

17 HOST Can be. - Bacteria: Gram (-) E. coli, gram (+) - Eukaryotic: yeast (Saccharomyces cerevisiae) - Mammalian: insect cells - Phage: filamentous phage Depends on - type of vector - post-translational modification

18 Filamentous phage genome, their products, and phage life cycle F pilus Tol A thioredoxin pi pxi piv ds DNA ss DNA II/X V VII IX VIII III VI I/IX IV replication virion assembly/export Illustrated by Dr. Santi Maneewatchararangsri

19 DNA cloning 1. Identification& selection of target DNA sequence/gene 2. Preparation of target DNA sequence/gene (coding) 3. Cloning of target DNA sequence to storage system o Verify inserted fragment by sequencing (Next)

20 DNA cloning 4. Selection of positive DNA sequence 5. Subcloning of the target DNA to expression system 6. Selection of positive DNA clone 7. Further experiments/ applications

21 Identification& selection of target DNA sequence/gene literature/ database in-silico prediction Bioinformatic tools Experiments Arrays (DNA/ RNA/ protein) Immuno/serology

22 Bioinformatic tools Prediction of functional protein association ParB MetK Transketolase Uncharacterized protein

23 Reference: Arnutti P, Kotepui M, Asanprakit W, Punyarit P, Petmitr PC, Harnroongroj T, et al. Determination of whole transcription profiles and specific pathways in invasive ductal breast carcinoma. Int J Clin Exp Pathol. 2013; 6(6):

24 Hierarchical cluster analysis of 19 IDC and 7 normal samples, according to 928 differentially expressed genes (p-value < 0.05 with 2-fold change). Arnutti P, et al. (2013)

25 Hierarchical cluster analysis of 19 IDC and 7 normal samples, according to 928 differentially expressed genes (p-value < 0.05 with 2-fold change). Arnutti P, et al. (2013)

26 Determination of gene expression profiles of selected genes by realtime RT-PCR. The ACTB gene in the tumors and normal tissue was quantified as the control gene copy number. Arnutti P, et al. (2013)

27 Experiments Reference: Reamtong O, Srimuang K, Saralamba N, Sangvanich P, Day NP, White NJ, et al. Protein profiling of mefloquine resistant Plasmodium falciparum using mass spectrometry-based proteomics. Int J Mass Spectrum. 2015; 391: Available from:

28 Coomassie blue stained gels of mefloquine-sensitive and mefloquine resistant P. falciparum proteins separated by SDS-PAGE. Reamtong et al. (2015)

29 Up-regulated proteins in mefloquine-sensitive and mefloquine-resistant P. falciparum. Reamtong et al. (2015)

30 Reference: Maneewatch S, Sakolvaree Y, Saengjaruk P, Srimanote P, Tapchaisri P, Tongtawe P, et al. Monoclonal antibodies to LipL32 protect against heterologous Leptospira spp. challenge. Hybridoma (Larchmt). 2008; 27(6): Available from: doi: /hyb

31 Proteome (A) and 2DE-immunoblotting of L. borgpetersenii probed with mablpf1 (B) and mablpf2 (C) Maneewatch S, et al. (2008)

32 Preparation of target DNA sequence/gene (coding) Type of Genetic materials: bacteria, virus, mammalian DNA RNA Plasmid Prokaryotic vs Eukaryotic : exon, intron Technology to amplify/ synthesize DNA

33 TECHNOLOGY Polymerase chain reaction (PCR): - can apply to unknown sequence - Library construction (for diversity of gene) - need optimization of PCR condition Interested gene Synthesize oligonucleotide: - Easy - Specific sequence

34 Cloning of target DNA sequence to storage system AAA TTT AAA TTT amplicons amplified by Taq DNA polymerase contain d(a) at both ends of the products they could be hybridized and ligated with d(t) overhanged vector

35 Cloning of target DNA sequence to storage system AAA AAA DNA LIGATION TTT TTT blunt/cohesive ATP: cofactor T4 DNA ligase

36 Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA Enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids Joins DNA fragments with either cohesive or blunt termini no activity on single-stranded nucleic acids.

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43 Selection of positive DNA sequence Colony: drug resistant E. coli Blue/white colony: beta galactosidase Fluorescense colony: GFP-tag Molecular methods - PCR - DNA sequencing

44 Subcloning of the target DNA to expression system Restriction site: compatible of insert DNA and linearlized vector Ligation : sticky end, blunt end Type of Vector Enzymes used in Molecular Biology

45 Characterization/ applications DNA sequencing Phenotype screening: Blue/white/ fluorescence colony Recombinant protein expression Molecular methods: PCR, RFLP, etc.

46 Characterization/ applications Recombinant protein expression

47 Characterization/ applications Molecular methods: PCR-RFLP of ScFv antibody library CDR1 CDR3 CDR2

48 End of session