Transcript Slajd 1
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: 17-28nt 2. 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. • When placed in an electrical field, DNA will migrate toward the positive pole (anode). • An agarose gel is used to slow the movement of DNA and separate by size. H O2 DNA - Power + 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 DNA small large - Power + 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. Electrophoresis Equipment Power supply Cover Gel tank Electrical leads Casting tray Gel combs PCR modifications: 1 – Nested PCR 2 –Touch-down PCR 3 – RT-PCR 4 – Real-time PCR Applications of the PCR 1 – Detection of the polymorphisms 2 – Diagnostics of hereditary diseases 3 – Sequencing (detection of mutations, paternity tests) 4 – Detection of viruses, parasites and bacteria 5 – Detection of GMOs 6 – In situ PCR (detection of given sequences in given subcellular localizations) 7 – Estimation of gene expression level In situ amplification (In situ PCR) Proteinase K Formalin paraformaldehyde Denhardt’s solution Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) The most sensitive technique for mRNA detection This task is integral to cloning complementary DNAs (cDNAs), which are DNA copies of mature mRNA. 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 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 der #9 #22 ph1 bcr bcr abl abl Translocation RT-PCR for bcr-abl variable length DNA transcription nuclear RNA RNA processing (in nucleus) mRNA abl primer bcr primer cDNA Reverse Transcriptase (RT) } PCR RT-PCR for bcr-abl M P C+ C- Multiplex PCR Identical (clonal) rearrangement of immunoglobulin (Ig) and/or T cell receptor (TCR) genes is observed in majority of lymphatic malignant tumors. TCRG genes rearrange in early stages of T cell differentiation Combinatorial repertoire for TCRG/D: ~5x103 for TCRA/B: ~3x106 and ~2x106 for Ig. Real-Time PCR (Q-PCR) R : reporter TaqMan® Method Q : quencher 3. intensifier 1. halogen tungsten lamp 2b. emission filters 2a. excitation filters 4. sample plate 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 Normal Extract RNA Extract RNA Tumor Make cDNA Amplify by PCR PCR Product Labeled with Green Dye PCR Product Labeled with Red Dye Mix Hybridize on Green Signal RNA Expressed in Normal Tissue Micro Array Red Signal RNA Expressed in 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 antihuman) 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) Results Group 1 Group 2 Group 3 Group 4 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? Approaches: a) Make antibodies - immunofluorescence b) “Express” the protein in cells with a tag Fuse to GFP 2) What is it doing in the cell? Approaches: a) Reduce protein levels - RNA interference b) 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 Virally-mediated introduction of the DNA Infection: Use recombinant viruses to deliver DNA Retroviruses Adenoviruses Carrier-mediated introduction of the DNA Positively charged carrier molecules are mixed with the DNA and added to cell culture media: Calcium Phosphate Dextran liposomes Carrier-DNA complexes bind to plasma membrane and are taken up DNA “expression” vector transfected: For expression in cells Insert gene in here Polyadenylation site To generate stable cell line pCMV/GFP For amplification of the plasmid in bacteria pUC Polyadenylation site Bacterial origin of replication 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