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Basic molecular biology and genetic engineering The central dogma DNA RNA Protein The big three molecules for genetics • DNA (DexoyribioNucelic Acid) – Heredity genetic information of an individual – Encode protein sequences (“genetic code”) • RNA (RiboNucleic Acid) – Transfer short pieces of information to cytoplasm – Provide templates to synthesize protein • Protein – – – – Produced via “translation” of messenger RNA (mRNA) Each protein has one or more specific functions Form body’s major components Carbohydrate and lipid metabolism mediated by proteins Related terminology • Genome – an organism’s complete set of DNA. • Gene – basic physical and functional units of heredity. – specific sequences of DNA encode instructions on how to make proteins. • Chromosome – an organized structure of DNA and protein in the nucleus. – contains many genes and regulatory elements • Gene expression – protein product is being made via transcription and translation The central dogma •Proposed by Francis Crick in 1958 to describe the flow of information in a cell. DNA •Information stored in DNA is transferred residue-byresidue to RNA which in turn transfers the information residue-by-residue to protein. RNA Protein •It has undergone numerous revisions. The Central Dogma Replication duplication of DNA using DNA as the template DNA Transcription synthesis of RNA using DNA as the template RNA Translation synthesis of proteins using RNA as the template Protein DNA & RNA Chains The building blocks for DNA and RNA DNA Adenine (A) Cytosine (C) Guanine (G) Thymine (T) RNA Adenine (A) Cytosine (C) Guanine (G) Uracil (U) DNA: The Basis of Life • Deoxyribonucleic Acid (DNA) – Double stranded with complementary strands A-T, C-G • DNA is a polymer – Sugar-Phosphate-Base – Bases held together by H bonding to the opposite strand Chargaff’s rule A=T(U) and G=C Semi-conservative DNA replication Daughter DNA molecules contain one parental strand and one newly-replicated strand DNA polymerase Residue-by-residue information transfer (DNA) ATGAGTAACGCG TACTCATTGCGC DNA transcription (RNA) RNA AUGAGUAACGCG codon translation (protein) Met-Ser-Asn-Ala Protein replication ATGAGTAACGCG TACTCATTGCGC + ATGAGTAACGCG TACTCATTGCGC Codon: The sequence of 3 nucleotides in DNA/RNA that encodes for a specific amino acid. Human genome are completely sequenced at 2003. What exactly does that mean? Overview of organizations of life • • • • • Nucleus = bookshelf Chromosomes = books Genes = words Nucleic acid = letters We are still learning the meaning of the words – apple = - zxuriguhwefrhqjrnfg =? – Zombie = - manzano =? We know only small part of protein function Any genome Similar Known Unknown Transcription DNA RNA Protein • Making messenger RNA (mRNA) from part of DNA by RNA polymerase • Needs a promoter region to begin transcription. Transcription closed promoter complex Transcription factors + RNA polymerase open promoter complex initiation elongation termination mRNA Where to start Transcription? Different cells have different gene transcription pattern Promoter: A specific DNA sequence for RNA polymerase and transcription factors binding Different transcription factors recognizes different promoters Exon and intron EXON: In protein coding genes, the segment of a gene that consists of a sequence of nucleotides that will be eventually translated into protein. INTRON: Non coding region of eukaryotic gene (transcribed into RNA than spliced) DNA Annotation of eukaryotic genomes Genomic DNA transcription Unprocessed RNA RNA processing Mature mRNA Gm3 AAAAAAA translation Nascent polypeptide folding Active enzyme Function Reactant A Product B Translation tRNA protein mRNA Ribosome Biologic Roles of RNA Messenger RNA (mRNA) carries information from DNA to the ribosomes, and is used as template to synthesize protein. Transfer RNA (tRNA) is a small RNA that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site according to the sequence of a bound mRNA. Ribosome synthesizes polypeptides under the direction of mRNA Mechanism of RNA interference (RNAi): post-transcriptional gene silencing shRNA, dsRNA What is RNA interference (RNAi)? • RNAi – ability of dsRNA to silence gene expression in a sequence-specific manner • Induced by short interfering (si)RNAs • Mechanism: siRNA-directed cleavage of mRNA via RISC complex Analyzing and manipulating a gene or genome DNA or RNA blotting What do we actually see? • Fluorescent chemistries-measurement by either: – Non-specific (SYBR green/EtBr) • Pros: inexpensive, no probe design • Cons: it reports ALL dsDNA formation not just the amplicon – Specific (probe) • Pros: increases specificity of the reaction, multiplex and mutation detection options • Cons: A bit more expensive, takes time to design probes, need a unique probe for each target Hybridization • Single-stranded DNA or RNA will naturally bind to complementary strands. DNA ATGAGTAACGCG TACTCATTGCGC RNA ATGAGTAACGCG UACUCAUUGCGC Probe hybridization Probe: a single-stranded DNA to detect the presence of a complementary sequence among a mixture of other singledstranded DNA or RNA DNA Microarrays Labeled DNA/RNA mixture flushed over array of short DNA fragments Laser activation of fluorescent labels DNA Microarray • measuring the amount of mRNA bound to each probe on the array. Affymetrix array Each spot (~ 100um) indicates a probe Overview GREEN represents Control DNA RED represents Sample DNA YELLOW represents a combination of Control and Sample DNA BLACK represents areas where neither the Control nor Sample DNA . Polymerase Chain Reaction • Goal: to amplify a low level DNA template for further analysis or manipulation • Need : – Primer: A set of single stranded DNA hybridize to the both end of target region – Taq polymerase: a thermostable DNA polymerase – Template DNA Is PCR quantitative? -A linear increase follows exponential -Eventually plateaus Taq polymerase has a half-life of 30 min at 95oC Log Target DNA Theoretical Reality Cycle # Real-time PCR Through the use of fluorescent molecules, real-time PCR allows us to ‘see’ the exponential phase so we can calculate how much we started with. 50-0.005 ng of template- FV Leiden primers The least the most DNA binding dyes 5’ 3’ Annealing 5’ 3’ 3’ BD 5’ Taq 3’ BD BD BD BD Taq Taq BD BD 5’ Extension BD 3’ BD BD Repeat Taq 5’ 3’ Extension continued apply excitation wavelength Fluorescence detection Emission Filter Excitation Filter Light Source Detector What does “cloning” mean? Clone: a collection of molecules, cells or individuals, all identical to an original one • • • • To "clone a gene" is to make many copies of it Gene can be an exact copy of a natural gene Gene can be an altered version of a natural gene Recombinant DNA technology makes it possible Recombinant DNA • taking a piece of one DNA, and combining it with another strand of DNA that would not normally occur together. • In order to do so, we need to • Copy it (PCR) • Cut it (restriction enzyme digestion) • Paste it (ligation) E. coli vector Human gene Restriction Enzymes – They cut the DNA at specific sequence. – Different restriction enzymes have different recognition sequences. Pasting DNA • Two pieces of DNA can be fused together by adding DNA ligase – Hybridization – complementary base-pairing – Ligation – fixing bonds with single strands Recombinant DNA Techniques Genetic Engineering • To transport a specific segment of DNA from one organism to another Put human gene to E.coli Introducing foreign into organism • The process whereby a DNA sequences are introduced by biologial, biochemical or physical processes. Transformation - bacteria Transfection – cell culture or virus Transgenesis – animal • Biological: virus infection • Biochemical: DEAE-dextran, calcium phosphate, and liposome-mediated transfection methods. • Physical: direct micro-injection of materials, biolistic particle delivery and electroporation. The Power of Recombinant DNA Technology – Human Insulin Production by Bacteria Human Insulin Production by Bacteria and cut with a restriction enzyme 6) join the plasmid and human fragment Human Insulin Production by Bacteria Mix the recombinant plasmid with bacteria. Human Insulin Production by Bacteria fermentor Transgenesis The stable integration of a foreign DNA into a host genome • DNA integrated into the genome • Germline transmission History of Transgenic mouse 1980-81:Several groups (Gordon et al., Brinster, Constantini et al, Lacy et al.) reported the development of transgenic mice by microinjecting genes into the pronucleus of a fertilized egg. 1982: The first visible phenotype was shown by Dr. R.Palmiter and colleagues in mice overexpressing rat growth hormone. A photograph of a giant transgenic mouse was on the cover of Nature. Transgenic mice Foreign DNA introduced into the germ line of mice by pronuclear injection; random gene insertion - specific genes to study pathogenesis of the disease - gene expressed using viral promoters/cell specific promoter Promoter gene pA Mouse Transgenesis Methods pros cons Relatively simple and efficient Long transgenes possible Potentially all species Random integration Multicopy insertions Strain limitations labor intensive, time consuming Very efficient Single copy insertions No technical equipment Works in many species High embryo mortality Expression level 9.5 kb packaging limit Safety issues (?) Pronuclear microinjection Lentivral infection Transgene DNA founder What is a Knockout Mouse? • A mouse in which a very specific endogenous gene has been altered in such a way that interferes with normal expression, i.e. it has been knocked out. Gene knockout overview Analysis in human