course contents dna and rna isolation quantification of dna and rna primer designing pcr

21
Course Contents 1. DNA and RNA isolation 2. Quantification of DNA and RNA 3. Primer designing 4. PCR 5. Electrophoresis 6. Sequencing 7. Karyotyping 8. Restriction Mapping 9. Flow cytometry 10.Hybridization a. Western blotting b. Southern blotting c. Northern blotting d. FISH

Upload: sanura

Post on 24-Feb-2016

63 views

Category:

Documents


0 download

DESCRIPTION

Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR Electrophoresis Sequencing Karyotyping Restriction Mapping. Flow cytometry Hybridization Western blotting Southern blotting Northern blotting FISH. Tissue culturing - PowerPoint PPT Presentation

TRANSCRIPT

Page 1: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Course Contents1. DNA and RNA isolation2. Quantification of DNA and RNA3. Primer designing4. PCR5. Electrophoresis6. Sequencing7. Karyotyping8. Restriction Mapping

9. Flow cytometry10.Hybridization

a. Western blottingb. Southern blottingc. Northern blotting d. FISH

Page 2: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

11.Transfection

12.Transduction

13.Transformation

14.Cloning

15.Microarrays

16.Chromatography

17.Immunochemistry

18.ELISA

19.Bioinformatics and techniques

20.Ethical issues

21.Tissue culturing

22.Slide Preparation and Cell Stains

23.Agar plate preparation and streaking for

the purpose of individual colony isolation

24.Bacterial Growth on selective agar

25.Quantification: Colony Forming Units

(CFU)

26.Dilution Plating

27.Identification and characteristics of

colonies

Page 3: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Assignment

Presentation

Quiz

Page 4: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Transfection :

is the process of deliberately introducing nucleic acids into cells.

Transformation:

non-viral DNA transfer in bacteria, non-animal eukaryotic cells and plant cells.

Transduction: is often used to describe virus-mediated DNA transfer.

What to transferGenetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected.

Target:Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material.

Page 5: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

TRANSFECTION  Transient TransfectionRapid, scalable, high-yield protein production from transiently transfected suspension cultures.the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded.

Stable TransfectionStable transfection introduces DNA into cells long-term and pass the introduced DNA to their progeny.

marker gene is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin

Page 6: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Gene name Gene product Assay

LacZ β-galactosidase enzyme assay or Histochemical

cat Chloramphenicol acetyltransferase

Chloramphenicol resistant

neo Neomycin phosphotransferase G418 resistant

gfp Green fluorescent protein Fluorescent

rfp Red fluorescent protein

microscopical, spectrophotometry

Common Marker Genes 

Page 7: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

 calcium phosphate, HEPES((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid )-buffered

saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution

containing the DNA to be transfected. When the two are combined, a fine precipitate of the

positively charged calcium and the negatively charged phosphate will form, binding the DNA

to be transfected on its surface. The suspension of the precipitate is then added to the cells to be

transfected (usually a cell culture grown in a monolayer). By a process not entirely understood,

the cells take up some of the precipitate, and with it, the DNA.

CHEMICAL-BASED TRANSFECTION

Page 8: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

• highly branched organic compounds, so-called dendrimers, to bind

the DNA and get it into the cell.

Page 9: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

•Liposomes: small, membrane-bounded bodies that are in some ways similar to the

structure of a cell and can actually fuse with the cell membrane, releasing the DNA into

the cell.

Liposomes can be created by sonicating phosphatidylcholine rich phospholipids in water.

Low shear rates create multilamellar liposomes, which have many layers like an onion.

Continued high-shear sonication tends to form smaller unilamellar liposomes. In this

technique, the liposome contents are the same as the contents of the aqueous phase.

Sonication is generally considered a "gross" method of preparation as it can damage the

structure of the drug to be encapsulated.

Page 10: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

•Electroporation transient increase in the permeability of cell membrane is achieved

when the cells are exposed to short pulses of an intense electric field.

Sonoporation uses high-intensity ultrasound to induce pore formation in cell membranes.

•Optical transfection is a method where a tiny (~1 µm diameter) hole is transiently

generated in the plasma membrane of a cell using a highly focused laser.

•Protoplast fusion is a technique in which transformed bacterial cells are treated with

lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai

virus, PEG, or electroporation) are used in order to fuse the protoplast carrying the gene of

interest with the target recipient cell. A major disadvantage of this method is that bacterial

components are non-specifically introduced into the target cell as well.

•Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is

inserted into a cell. This approach can also be implemented with arrays of nanofibers that

are introduced into large numbers of cells and intact tissue.

NON-CHEMICAL TRANSFECTION

Page 11: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

• GENE GUN: where the DNA is coupled to a nanoparticle of an inert solid

(commonly gold) which is then "shot" directly into the target cell's nucleus.

PARTICLE BASED TRANSFECTION

Page 12: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Magnetofection, Nucleic acids are first associated with magnetic nanoparticles.

Then, application of magnetic force drives the nucleic acid particle complexes towards

and into the target cells, where the cargo is released.

Page 13: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Impalefection is carried out by impaling cells by elongated nanostructures and

arrays of such nanostructures such as carbon nanofibers or silicon nanowires which

have been functionalized with plasmid DNA.

•particle bombardment. The nucleic acid is delivered through membrane penetration at

a high velocity, usually connected to micro projectiles.

Page 14: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

TRANSDUCTION 

Introduced of DNA into cells using viruses as a carrier

Page 15: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Viral vectors are tailored to their specific applications but generally share a few key

properties.

Safety: modified in such a way as to minimize the risk of handling them by deletion of a part of the

viral genome critical for viral replication.

Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it

infects.

Stability: Some viruses are genetically unstable and can rapidly rearrange their

genomes. This is detrimental to predictability and reproducibility of the work conducted using

a viral vector and is avoided in their design.

Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell

types as possible. However, sometimes the opposite is preferred. The viral receptor can be

modified to target the virus to a specific kind of cell. Viruses modified in this manner are said

to be pseudo typed.

Identification: Viral vectors are often given certain genes that help identify which cells

took up the viral genes.

Page 16: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Basic research

Gene therapy

vaccines

APPLICATIONS

Page 17: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

Immortalization of primary T cells

GP2-293

GP2xTERT11 PRODUCER CELL LINE ENVELOPE CONSTRUCT

PRIMARY T CELLS

PACKAGED TERT RECOMBINANT VECTOR

N S

Staining with anti-NGFR ab beads

Page 18: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

number of FDA-approved clinical trials such as the SCID-X1 trial.

either be replication-competent or replication-defective..

involves the requirement for cells to be actively dividing for transduction.

cells such as neurons are very resistant to infection and transduction by

retroviruses.

There is concern that insertional mutagenesis due to integration into the

host genome might lead to cancer or leukemia

Retroviruses

Page 19: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

LENTIVIRUSES

Lentiviruses are a subclass of Retroviruses.

able to integrate into the genome of non-dividing cells, The site of integration is

unpredictable,

studies have shown that lentivirus vectors have a lower tendency to integrate in

places that potentially cause cancer , clinical trials that utilized lentiviral vectors to

deliver gene therapy for the treatment of HIV experienced no increase in mutagenic

or oncologic events.

One or more plasmids, generally referred to as packaging plasmids, encode

the virion proteins, such as the capsid and the reverse transcriptase. Another plasmid

contains the genetic material to be delivered by the vector. It is transcribed to

produce the single-stranded RNA viral genome and is marked by the presence of

the ψ (psi) sequence. This sequence is used to package the genome into the virion.

Page 20: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

ADENOVIRUSES

As opposed to Lentiviruses, adenoviral DNA does not integrate into the

genome and is not replicated during cell division.

with adenoviruses, which cause respiratory, gastrointestinal and eye infections,

they trigger a rapid immune response with potentially dangerous consequences.

To overcome this problem scientists are currently investigating adenovirusesto

which humans do not have immunity.

Page 21: Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

ADENO-ASSOCIATED VIRUSES

AAV is not currently known to cause disease and consequently the virus

causes a very mild immune response. AAV can infect both dividing and

non-dividing cells and may incorporate its genome into that of the host

cell. These features make AAV a very attractive candidate for creating viral

vectors for gene therapy