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Molecular Biotechnology
In the name of God Molecular Biotechnology By: Mohsen Naeemipour
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References Molecular biotechnology : principles and applications of recombinant DNA by Bernard Glick, Jack Pasternak An introduction to molecular biotechnology : molecular fundamentals, methods and applications in modern biotechnology By Wink Michael
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Timetable of Course The Development of Molecular Biotechnology
DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals
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Lecture 1: The Development of Molecular Biotechnology
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Many scientific disciplines contribute to molecular biotechnology, which generates a wide range of commercial products 5
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Microbial Biotechnology
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Biology systems in biotechnology
Prokaryote (E.Coli, B.Subtilis) Eukaryote (S. Cerevisiae, CHO)
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Microbial Biotechnology
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Lecture 2: DNA, RNA and Protein
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Timetable of Course The Development of Molecular Biotechnology
DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Bioinformatics, Genomics, and Proteomics Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Transgenic Animals
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DNA Replication 13
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Model of an SV40 DNA replication fork
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DNA Repair 22
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Proofreading by DNA polymeras
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Deamination leads to point mutation
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Base excision repair January 14, 2019
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Mismatch excision repair in human cell
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Nucleotide excision repair in human cells
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Some Inherited Syndromes with Defects in DNA Repair
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Transcription and translation
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Types of RNA Polymeras RNA Pol l (rRNA 5.8s, 18s. 28s)
RNA Pol ll (mRNA, miRNAs, snRNAs) RNA Pol lll (tRNA, 5s rRNA) January 14, 2019
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Structure of the 5' methylated cap
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Overview of RNA processing
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Alternative splicing January 14, 2019
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Prokaryotic and eukaryotic ribosome components
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initiation of translation in eukaryotes
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initiation of translation in eukaryotes
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Peptidyl chain elongation in eukaryote
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January 14, 2019
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Termination of translation in eukaryote
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Multiple reading frames in an mRNA sequences
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Control of Gene Expression
in prokaryotes 44
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Regulation of transcription from the lac operon of E.coli
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Lac repressor-operator interaction
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Sigma Factors of E.coli January 14, 2019
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DNA looping permits interaction of bound NtrC and RNA polymerase
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The PhoR/PhoB two component regulatory system in E. coli.
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Timetable of Course The Development of Molecular Biotechnology
DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Bioinformatics, Genomics, and Proteomics Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Transgenic Animals
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Recombinant DNA Technology
Lecture 3: Recombinant DNA Technology
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Restriction Endonucleases
Isoschizomers (XhoI and PaeR7I) Neoschizomers (NarI and KasI) Methylation status of DNA (HpaII, MspI)
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Partial digestion of a fragment of DNA
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Restriction endonuclease digestions and electrophoretic separation of fragments
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Plasmid Vectors High-copy-number plasmids (10 to 100 copies)
Low-copy-number plasmids (1 to 4 copies) Incompatibility group Narrow- and broad-host-range plasmids
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Replica plate January 14, 2019
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Some antibiotics commonly used as selective agents
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Insert capacities of some commonly used vector systems
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Genomic library January 14, 2019
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Screening a library with a labeled DNA probe (colony hybridization)
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Immunological screening of a gene library (colony immunoassay)
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Gene cloning by functional complementation
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Separation of polyadenylated mRNA from total cellular RNA
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Genetic Transformation of Prokaryotes
Transferring DNA into E. coli Electroporation Conjugation
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Electroporation January 14, 2019
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A B C January 14, 2019
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Some of the enzymes used for recombinant DNA technology
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Timetable of Course The Development of Molecular Biotechnology
DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals
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Chemical Synthesis, Amplification, and Sequencing of DNA
Lecture 4: Chemical Synthesis, Amplification, and Sequencing of DNA
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Chemical Synthesis of DNA
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Chemical Synthesis of DNA
Assembling whole genes or parts of genes Amplifying specific DNA sequences Introducing mutations into cloned genes Screening gene libraries Sequencing DNA Facilitating gene cloning
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Flowchart for the chemical synthesis of DNA oligonucleotides
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Starting complex for the chemical synthesis of a DNA strand
Spacer molecule controlled-pore glass (CPG) bead, dimethoxytrityl (DMT) January 14, 2019
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A Phosphoramidite Molecular
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An anhydrous reagent (acetonitrile) Argon
Trichloroacetic acid (TCA) (Detritylation) Acetonitrile January 14, 2019
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Activation and coupling
phosphoramidite tetrazole January 14, 2019
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Acetic anhydride and dimethylaminopyridine are added
to acetylate the unreacted 5′ hydroxyl groups January 14, 2019
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The phosphite triester is oxidized with an iodine mixture to form the more stable pentavalent phosphate triester January 14, 2019
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Flowchart for the chemical synthesis of DNA oligonucleotides
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Overall yields of chemically synthesized oligonucleotides with
different coupling efficiencies January 14, 2019
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Uses of Synthesized Oligonucleotides
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Typical linker and adaptor sequences
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Cloning with a linker January 14, 2019
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Creating a restriction endonuclease site in a vector with an adaptor
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Enzymatic DNA synthesis of a gene
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Assembly of a synthetic gene from short oligonucleotides
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Assembly and in vitro enzymatic DNA synthesis of a gene
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Gene Synthesis by PCR January 14, 2019
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DNA-Sequencing Techniques
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Blocked DNA synthesis January 14, 2019
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B. deoxyribonucleotide
A. dideoxynucleotide B. deoxyribonucleotide January 14, 2019
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Primer extension during DNA synthesis in the presence of dideoxynucleotides
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Simulated autoradiograph of a dideoxynucleotide DNA-sequencing gel
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Automated fluorescent-dye terminator Sanger DNA sequencing
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DNA sequencing by primer walking
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Timetable of Course The Development of Molecular Biotechnology
DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals
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Expression in Prokaryotes
Lecture 5: Manipulation of Gene Expression in Prokaryotes
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Manipulation of Gene Expression
Promoter and transcription terminator sequences Strength of the ribosome-binding site Number of copies of the cloned gene Gene is plasmid borne or integrated into the genome of the host cell Final cellular location of the synthesized foreign protein Efficiency of translation in the host organism Intrinsic stability within the host cell of the protein encoded by the cloned gene. January 14, 2019
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Regulatable Promoters
lac and trp (tryptophan) operons promoters are commonly used hybrid constructs the ratio of the number of repressor protein molecules to the number of copies of the promoter sequences two different plasmids repressor gene is placed on a low-copy-number (1-8) high-copy-number plasmid (30-100)
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Regulation of gene expression controlled by the pL promoter
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A portion of the DNA sequence of the E
A portion of the DNA sequence of the E. coli lac promoter (plac) and its mutated, more active, form (pmut).
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pCP3 vector pPLc2833 plasmid + pKN402 Increasing Protein Production
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Large-Scale Systems Dual-plasmid system for controlling the λ pL promoter by regulating the cI repressor with tryptophan January 14, 2019
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Uses of Fusion Proteins
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