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實驗神經概論
9月13日:鄭菡若老師(Basic molecular biology and genetic engineering)
9月20日:鄭菡若老師(Protein science)9月27日:蔡金吾老師(Basic principles of microscopy)10月4日:楊定一老師(Basic cell biology in nervous
systems)10月11日:楊靜修老師(Introduction to neurophysiology
and sleep research)10月18日:洪成志老師(Basic behavioral science: animal
models of psychiatric disorders)
Basic molecular biology and genetic engineering
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The building structure of life
Genome
Chromosome
GeneNucleotide
• 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
• Genome
– an organism’s complete set of genetic information.
The central dogma
RNA
Protein
DNA
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The central dogma
RNA
Protein
DNA
•Proposed by Francis Crick in 1958 to describe the flow of information in a cell.
•Information stored in DNA is transferred residue-by-residue to RNA which in turn transfers the information residue-by-residue to protein.
•Such information cannot be transferred back from protein to either protein or nucleic acid.
•It has undergone numerous revisions.
The big three molecules for genetics
• DNA (DexoyribioNucelic Acid)
– Heredity genetic information of an individual
– Encode protein sequences (“genetic code”)
• RNA (RiboNucleic Acid)
– Transcribe 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
RNA
Protein
DNA
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The Central Dogma
Transcription
Translation
Replication
RNA
Protein
DNA
Duplication of DNA using DNA as the templateRequire: DNA polymerase
Synthesis of RNA using DNA as the templateRequire: RNA polymerase
Synthesis of proteins using RNA as the templateRequire: Ribosome
Nucleotides
– Sugar-Phosphate-Base
– The building block of DNA or RNA
Base
Sugar
Acid
3’
5’
1
2
3
4
5
6
Adenine (A)Guanine (G)Thymine (T)Cytosine (C)
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DNA & RNA Polymer Chains
Both DNA and RNA can synthesis only from 5’ 3’
DNAAdenine (A)Cytosine (C)Guanine (G)Thymine (T)
RNAAdenine (A)Cytosine (C)Guanine (G)Uracil (U)
The bases for DNA and RNA
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Rule for base pairingA-T and G-C
RNA
Protein
DNA
5’ATGAGTAACGCG3’3’TACTCATTGCGC5’
5’ATGAGTAACGCG3’3’TACTCATTGCGC5’
5’ATGAGTAACGCG3’3’TACTCATTGCGC5’
+
5’AUGAGUAACGCG3’
Met-Ser-Asn-Ala
transcription replication
(RNA)
(protein)
(DNA)
translation
codon
Residue-by-residue information transfer
Codon: The sequence of 3 nucleotides in DNA/RNA that encodes for a specific amino acid.
DNA polymerase
RNA polymerase
Ribosome
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Semi-conservative DNA replication
Daughter DNA molecules contain one parental strand and one newly-replicated strand
DNA polymeraseRNA
Protein
DNA
http://www.youtube.com/watch?v=z685FFqmrpo&feature=related
Transcription
• Making messenger RNA (mRNA) from part of DNA by RNA polymerase
• Needs a promoter region to control transcription.
RNA
Protein
DNA
Rule for base pairingA=U and G=C
http://www.youtube.com/watch?v=ztPkv7wc3yU&feature=related
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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
Where to start transcription?
Transcription
Transcription factors + RNA polymerase
closed promoter complex
open promoter complex
initiation
elongation
termination
mRNA
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EXON: In protein coding genes, the part of a gene that will beeventually translated into protein.
INTRON: Non coding region of eukaryotic gene (transcribed into RNA than spliced out)
Exon and intron in DNA and mRNA
DNA
Annotation of eukaryotic genomes
transcription
RNA processing
translation
AAAAAAA
Genomic DNA
Unprocessed RNA
Mature mRNA
Nascent polypeptidefolding
Reactant A Product BFunctional protein
Folded protein
Gm3
exon intron5’ 3’
5’ 3’
5’ 3’
N C
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Overview of organizations of life
• Nucleus = bookshelf
• Chromosomes = books
• Genes = words
• Nucleic acid = letters
– apple =
- manzano =?
Human genome are completely sequenced at 2003.What exactly does that mean?
Unknown
Similar
Known
•Only small portion of the genes have known function
•We need to learn the meaning of these sequences
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• Messenger RNA (mRNA) carries information from DNA to the ribosomes, sites of protein synthesis in the cell.
• Non-coding RNA is RNA that is not translated into a protein. Such as: tRNA, rRNA, shRNA, microRNA, siRNA….
– Transfer RNA (tRNA) transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation.
– MicroRNA (miRNA) functions in RNA silencing and post-transcriptional regulation of gene expression.
– Ribosomal RNA (rRNA) is a component of the ribosomes, and makes up at least 80% of the RNA molecules in a cell
Types of RNA
Translation
Ribosome
tRNA
mRNA
protein
RNA
Protein
DNA
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Transfer RNA
• Each type of amino acid has its own subset of tRNAs.• Each specific tRNA molecule contains a 3 nucleotide sequence,
an anticodon, that can base-pair with its complementary codonin the mRNA.
DNA Mutations
nonsense mutation
missense mutation
frameshift1-2 bps deletion
frameshift1-2 bps insertion
normal
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DNA
mRNA
mRNA
PROTEIN
Transcription
TransportandLocalization
Translation
Degradation
Processing
Protein activity(post-translation)
Control of eukaryotic gene expression
RNAi is an RNA-mediated control of gene expression
MicroRNA (miRNA)
• miRNAs are endogenous ~22 nt RNAs processed from longer RNA precursors
• They do not translate into protein
• Partial complementary binding to target mRNAs to regulate their stabilities
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RNA interference (RNAi):post-transcriptional gene silencing
shRNA, dsRNAmiRNA
• RNAi – ability of double stranded RNA to silence gene expression in a sequence-specific manner
• Induced by small interfering (si)RNAs
• Mechanism: siRNA-directed cleavage of mRNA via RISC complex
https://www.youtube.com/watch?v=cK-OGB1_ELE
Analyzing and manipulating a gene or genome
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What does “cloning” mean?
Clone: a collection of molecules, cells or individuals with identical genetic information to an original one
Molecular cloning: refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it
• To "clone a gene" is to make many copies of it
• 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)
Human gene
E. coli vector
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Copy- Polymerase Chain Reaction (PCR)
• 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
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Cut -Restriction Enzymes
– They cut the DNA at specific sequence.
– Different restriction enzymes have different recognition sequences.
Paste – DNA ligase
• Two pieces of DNA can be fused together by adding DNA ligase
– Hybridization –complementary base-pairing
– Ligation – fixing bonds with single strands
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Recombinant DNA Techniques
Genetic Engineering
• To transport a specific segment of DNA from one organism to another
Put human gene to E.coli
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Introducing foreign into organism
• The process whereby a DNA sequences are introduced by biologial, biochemical or physical processes.
Transformation - bacteriaTransfection – cell culture or virusTransgenesis – animal
• Methods:
• Biological: virus infection
• Biochemical: DEAE-dextran, calcium phosphate, and liposome-mediated transfection methods.
• Physical: direct micro-injection of materials, biolisticparticle delivery and electroporation.
The Power of Recombinant DNA Technology –Human Insulin Production by Bacteria
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Human Insulin Production by Bacteria
6) join the plasmid and human fragment
and cut with a restriction enzyme
Mix the recombinant plasmid with bacteria.
Human Insulin Production by Bacteria
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fermentor
Human Insulin Production by Bacteria
– Non-specific dyes (SYBR green/EtBr staining)
• Pros: inexpensive, no probe design
• Cons: it reports ALL dsDNA
– Specific probe (hybridization)
• 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
How can we actually detect DNA or RNA?
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Non-specific DNA staining
Specific sequence detection - Hybridization
• Single-stranded DNA or RNA will naturally bind to complementary strands.
5’ATGAGTAACGCG3’3’TACTCATTGCGC5’
5’ATGAGTAACGCG3’3’UACUCAUUGCGC5’
DNA
RNA
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DNA or RNA blotting(Southern ) (Northern )
Probe: a single-stranded DNA to detect the presence of a complementary sequence among a mixture of other singled-stranded DNA or RNA
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Probe hybridization
DNA Microarrays
Labeled DNA/RNA mixture flushed over array of short DNA fragments
Laser activation of fluorescent labels
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DNA Microarray
Affymetrix array
Each spot (~ 100um) indicates a probe
• measuring the amount of mRNA bound to each probe on the array.
DNA Microarray
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
.
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DNA sequence
RNA Sequencing (RNA-seq)
• uses next-generation sequencing to reveal the quantity of RNA in a biological sample at a given moment in time.
Normal
Tumor
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Is PCR quantitative?Is PCR quantitative?
Cycle #
Log
Targ
et
DN
A
Theoretical
-A linear increase follows exponential
Reality
-Eventually plateaus
Taq polymerase has a half-life of 30 min at 95oC
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.
Real-time PCR to compare RNA levelReal-time PCR to compare RNA level
50-0.005 ng of template- FV Leiden primers
the most
The least
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Detector
Fluorescence detectionFluorescence detection
EmissionFilter
Light Source
ExcitationFilter
Genetically modified mouse
• A mouse has its genome altered through the use of genetic engineering techniques.
• Genetically modified mice are commonly used for research or as animal models of human diseases.
• Two major types:– Add it in : Transgenic mouse
– Take it our: knockout mouse
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Transgenesis
The stable integration of a foreign DNA into a host genome
• DNA integrated into the genome• Germline transmission• For study the gain-of-function effect
1982: The first visible phenotype was shown in transgenic mice overexpressing rat growth hormone.
2009: first transgenic monkey were generated
History of transgenic animals
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What is a Knockout Mouse?
• Knock-out mouse - A mouse in which a very specific endogenous gene has been removed.
• For study loss-of-function effect
A B C
A C
DNA
DNA
Control of gene expression
PatternTemporal – inducible promoterSpatial – Tissue/cell type specific promoter
Level – inducible or house keeping gene
Tetracycline regulated system
Cre-mediated gene activation
Tamoxifen regulated system
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CamK2a GAD67GFAP
Tissue/cell type specific promoter
• Use tissue/cell type specific promoter• Allow the study of separate functions of
pleiotropic genes
Tetracyclin-dependent inducible system
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Basic principle:
gene
Tissue-specific promoter Cre
Target gene #1
Transgene #2
Cre-activated conditional knockout
loxP loxP
Floxed : Flanked by loxP (or FRT)sites.
“Two stage” strategies control gene expression in where and when
a. Cre-recombinase with a cell type-specific promoter plus marker gene
b. Transcriptional activators under pharmaceutical control
Tamoxifen inducible system
4-OH-tamoxifen – a fake estrogen – binds to estrogen receptor (ER).
Cre-ER
•a fusion of Cre with a mutant estrogen receptor that DOSE NOT binds estrogen but DOES bind tamoxifen.
•Normally complex with Hsp90 and stay in cytoplasm
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Cre-ER is activated after addition of tamoxifen
Floxed genein the nucleus
Complex of Cre-ER with Hsp90 In cytoplasm
No recombination
Cre-ER-TX dissociates from Hsp90, goes to nucleus, and removes floxed gene
NO TAMOXIFEN TAMOXIFEN added
Hsp90
Tamoxifen (TX)
Cre-ER
Application for genetic modified animals