Basic molecular biology and genetic engineering

9/12/2016

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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

9/12/2016

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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’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.


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fermentor


– 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’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

Documents

Basic molecular biology and genetic engineering