Molecular biology techniques

Bartosz Brzezicha

In a microtube :

- DNA to be amplified (cloned DNA, genomic DNA, RNA/cDNA)

- 2 primers

- dNTPs

- Taq Polymerase

- buffer with Mg++

Final volume : 20-100 l

Equipment: Thermocycler

General Considerations PCR primers

1. Primer length: 17-28nt2. Melting Temperature (Tm) for each primer = 50 –

65ºC.3. Difference between Tm of primers max. 5ºC.4. Primers should not contain 4 consecutive G/C

residues. The last nucleotide at the 3’-end of the primer should be C/G.

5. Optimize concentration of forward and reverse primers to be used

6. Primer self-complementarity (ability to form 2nd order structures such as hairpins) should be avoided

Rough estimation of melting temperature:

Tm = 4 * n(G/C) + 2 * m(A/T)

Is there a gene copied during PCR and is it the right size ?

Verification of the PCR product on gel.

• DNA is negatively charged.

+-

Power

DNA

• When placed in an electrical field, DNA will migrate toward the positive pole (anode).

H

O2

• An agarose gel is used to slow the movement of DNA and separate by size.

+-

Power

DNA

How fast will the DNA migrate?strength of the electrical field, buffer, density of agarose gel…

Size of the DNA!*Small DNA move faster than large DNA…gel electrophoresis separates DNA according to size

smalllarge

Staining the Gel• Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel.

• Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run.

Casting tray

Gel combs

Power supply

Gel tank Cover

Electrical leads

Electrophoresis Equipment

PCR modifications:

1 – Nested PCR2 –Touch-down PCR3 – RT-PCR4 – Real-time PCR

Applications of the PCR

1 – Detection of the polymorphisms2 – Diagnostics of hereditary diseases3 – Sequencing (detection of mutations, paternity tests)4 – Detection of viruses, parasites and bacteria5 – Detection of GMOs6 – In situ PCR (detection of given sequencesin given subcellular localizations)7 – Estimation of gene expression level

In situ amplification (In situ PCR)

Formalin paraformaldehyde

Denhardt’s solution

Proteinase K

The most sensitive technique for mRNA detection

Reverse transcriptase:a DNA polymerase that uses RNA as its template. Thus it is able to make genetic information flow in the reverse (RNA to DNA) of its normal direction

This task is integral to cloning complementary DNAs (cDNAs), which are DNA copies of mature mRNA.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

RT- PCR, Cont…

Detection of t(9;22) in CML

Detection of circulating blood cells expressing bcr-abl chimeric RNA following bone marrow transplant is an indicator of likely relapse.

RT-PCR for bcr-abl

#9 #22 #9 #22

abl

bcr

#9 der ph1

abl

bcr

Translocation

RT-PCR for bcr-abl

variable length

transcription

DNA

nuclear RNA

RNA processing (in nucleus)

mRNA

Reverse Transcriptase (RT)

cDNA

abl primer

bcr primer

} PCR

RT-PCR for bcr-abl

M P C+ C-

TCRG genes rearrange in early stages of T cell differentiation

Combinatorial repertoire for TCRG/D: ~5x103

for TCRA/B: ~3x106 and ~2x106 for Ig.

Identical (clonal) rearrangement of immunoglobulin (Ig) and/or T cell receptor (TCR) genes is observed in majority of lymphatic malignant tumors.

Multiplex PCR

Real-Time PCR (Q-PCR)TaqMan® Method

R : reporter

Q : quencher

2a. excitation filters

2b. emission filters

1. halogen tungsten lamp

4. sample plate

3. intensifier

5. ccd detector

Gene expression detection and its level estimation

Southern Hybridization Detects:

• Presence (or Absence) of a Gene

• Variation in the sequence of a Gene (Polymorphism)

• Increased copy number of a Gene (Gene Amplification)

• Gene rearrangement (Chromosomal Translocation)

Micro Arrays

• Test for the presence of a nucleic acid sequence by hybridizing a probe bound to a matrix to the target sequence.

• Many different probes can be bound to the same matrix.

• Therefore, a single sample can be evaluated for many different target sequences simultaneously.

Micro Arrays

• Expression Arrays - test for mRNA expressed in a tissue.

• Sequencing Arrays - test for nucleotide sequence in a fragment of DNA (sequencing by hybridization - ideal for detection of single nucleotide polymorphisms [snps]).

Performing a Microarray Study

NormalTumor

Extract RNA

Extract RNA

Make cDNAAmplify by PCR

PCR ProductLabeled withGreen Dye

PCR ProductLabeled withRed DyeMix

Hybridize on Micro Array

Green SignalRNA Expressedin Normal Tissue

Red SignalRNA Expressedin Tumor Tissue

Classification of Diffuse Large B-Cell Lymphoma by Microarrays

Alizadeh et al., Nature 403:503, 3 Feb 2000

SDS-PAGE• SDS is an ionic detergent that readily binds to protein and

gives it an overall negative charge. This allows the protein to travel towards the (+) charge, and the fragments separate according to its molecular weight as it travels.

• Staining/Blotting– Coomassie Blue stain binds to protein but not the

polyacrylamide, therefore it will only stain at sites where protein are present.

Procedures

• Prepare polyacrylamide gels (separating and stacking gels)

• Load protein samples into wells• Run gel• Analysis of results by staining with

Coomassie blue or Western Blot

Western Blot• Gel was nicked at one corner as

marker.• The gel was then placed into a

sandwich clamp used for western blotting:– Sponge– Filter paper– Gel– Nitrocellulose Membrane– Filter paper– Sponge

• Sandwich clamp was then placed into the transfer apparatus along with a block of ice and a stir bar in an ice container.

• The apparatus was filled with transfer buffer and the gel ran at 100 V for one hour while spinning.

Western Blot• Transfers protein from SDS-PAGE to the

nitrocellulose membrane.• TBST and 5% nonfat milk were used for

blocking nonspecific binding sites.• Membrane reacts with primary (ex. rabbit

anti-human) antibody for 1 hour• Wash with TBST to get rid of unbound

primary antibody• React membrane with secondary (ex.

donkey anti-rabbit) antibody conjugated to HRP.

• Wash and react with substrate (radiolabelled, fluorescent, or with colour)

ResultsGroup 1Group 1 Group 2Group 2

Group 4Group 4Group 3Group 3

Applications

• SDS-PAGE– Isolation of protein– Approximating the length of a polypeptide– Determining the molecular weight of protein

bands

• Western Blotting - used for Medical diagnosis– Detect HIV-antibody in human serum sample– Detect autoantibodies in human serum sample– Lyme disease

Exploring protein function

1) Where is it localized in the cell?

2) What is it doing in the cell?

Approaches:a) Make antibodies - immunofluorescence

b) “Express” the protein in cells with a tag Fuse to GFP

Approaches:a) Reduce protein levels - RNA

interferenceb) Increase protein levels “over-express”

c) “Express” mutant versions

Direct introduction of the DNA

Electroporation - electric field temporarily disrupts plasma membrane

Biolistics (gene gun)- fire DNA coated particles into cell

Microinjection

Biolistics Particle Delivery

Infection:Use recombinant viruses to deliver DNA

RetrovirusesAdenoviruses

Virally-mediated introduction of the DNA

Positively charged carrier molecules are mixed with the DNA and added to cell culture media:

Calcium PhosphateDextranliposomes

Carrier-DNA complexes bind to plasma membrane and are taken up

Carrier-mediated introduction of the DNA

DNA “expression” vector transfected:

pCMV/GFP

CMV

Promote

r

Insert genein here

Polyadenylationsite

SV40

Pro

mote

rN

eom

ycin

resista

nce

PolyadenylationsitepUC

Bacterial origin of replicationA

mpicillin

resista

nce

For expression in cells

To generate stable cell line

For amplification of the plasmid in bacteria

GFP

EXPERIMENT:

Transfect unknown-GFP fusion protein

Visualize GFP protein fluorescence by fluorescence microscopy in living cells

Counter-stain with known marker to compare localization patterns in living cells

= “vital stain”

Transformed cell:

• Transmitted light:

• Fluorescent light:

FISH vs. CISH

FISH -fluroscence in situ hybridization

CISH - chromogenic in situ hybridization

Tests to detect HER ( human epidermal growth factor receptor ) positivity in breast cancer

Detection of Increased Number of Her-2/neu Genes by Fluorescence In Situ Hybridization

(FISH)

Normal Amplified