Lecture 1MOLECULAR BIOLOGY

Introduction to Molecular Biology TechniquesDr. Jane Sherwood H1.18Email jms@dmu.ac.ukI work Mondays Tuesdays and Thursdays

Introduction to Molecular Biology Techniques What is molecular biology? What are its uses? Important background revision DNA/RNA/protein structure

TextbooksEssential Cell Biology (ECB)Chapter 10Alberts et al. Published by Garland

TextbooksAny other up-to-date (since 2009) molecular biology text book

Areas to be covered during the next 4 weeks (plus 4 weeks in 2016) Purification of nucleic acids Manipulating DNAPCR including qPCR Sequencing of DNA Sanger method and new generation sequencing Southern (Dot blots) and Northern blotting Western blottingFISH and chromosome painting

Learning objectives To review the structure of DNA and RNA To be able to draw flow diagrams to illustrate the processes of DNA and RNA purification To know how to quantify how much DNA or RNA there is in your sample

Central Dogma of Molecular BiologyFrom Essential Cell Biology Alberts et al.

From Essential Cell Biology Alberts et al.

From Essential Cell Biology Alberts et al.

Purification of DNAMany different commercial kits available

DNA free from RNA and _________ contamination

Purification/isolation of DNAFrom Principles and techniques of biochemistry and molecular biology byWilson and Walker 7th Ed CUPPre-kit method

Usually use commercially available kits but...you need to know the function of each reagent for troubleshooting when things go wrong!Wizard genomic DNA purification kit from whole bloodpure ds DNAPDF file for instructions

Blood storage prior to purification of DNACan be sent by first class postNeeds to be collected in anti-coagulant (stops blood clotting), ie. ________, citrate, heparin2-8 C for up to 2 months (?yields)

Obtaining the DNA from the white blood cellsCell lysis solutionNaOH and SDS (sodium dodecylsulphate)Lysis of red cells leaving white cells intactGentle inversion of tubeCentrifuge to remove ______ cell components - pellet = white cells

Nuclei lysis solutionWhite cells and their nuclei are lysed and solubilizedOptional RNase treatment at this stage (Southern blots)Debris in ________ - DNA in supernatant

Purifying the DNAProtein precipitationSalt precipitates cellular proteins leaving high molecular weight genomic DNA in solutionIsopropanol/ethanol precipitationGenomic DNA is concentrated and desalted

Store DNA at 2-8 C _________ 5-15g from 300l whole blood

From :Wizard Genomic DNA Purification KitAdd RNase here

Checking the integrity and quantity of DNAGel electrophoresisCheck integrityEstimate yield (quantity)Run DNA samples of known concentration and compared intensity of bands to unknown - rough estimate only!DNA determination by spectrophotometryAmount of UV absorbed by sample is directly proportional to amount of DNAAbsorbance260nm of 1.0 = 50 g of ds DNA/mL

RNAFrom Essential Cell Biology Alberts et al.

Isolation of RNAWhat type of RNA? Total RNA mRNA

Isolation of RNA Many different methods Problem RNases Present all around particularly on our skin Take care not to degrade the RNA you have isolated All glassware, equipment and water must be treated with DEPC (diethylpyrocarbonate)Inhibits RNase __________ Many commercial kits are available to isolate RNA

Isolation of RNA - general steps involvedFrom Principles and techniques of biochemistry and molecular biology byWilson and Walker 7th Ed CUP

Total RNA isolationThe PAXgene Blood RNA System from PreAnalytiX

From Patient to purified total RNA consistent results

Isolation of mRNA using magnetic particles

All mRNA molecules have a poly A tail (polyadenylation) Poly A tail will hybridize to poly T oligonucleotide Attach poly T to biotin (= biotinylated oligonucleotide) Biotin will tightly bind to streptavidin which is used to coat magnetic beads (Dynabeads)

TTTTTTTTTTTTTTTTTTTTTTPoly T tailAAAAAAAAAAAAA53mRNA

Mix with sample (cells/ nuclei broken open)polyT binds to poly A tail of mRNAMix with streptavidin paramagnetic particles (SA-PMPs)mRNA now bound to magnetic particlesUse magnet to form pellet Remove s/n (supernatant)Wash beads/mRNA with bufferThis washes away impurities leaving _______ mRNA bound to beads - release mRNA from poly T tail by resuspending pellet in water

Isolation of mRNA using magnetic particles

Advantages:Quick, pure (no contaminating DNA or proteins)Safer than chemically for traditional methodsDisadvantages:Expensive, only for mRNA (need poly A tail)Kits are also available for total RNA preparation from various sources

Quantification and integrity of RNARNA determination by spectrophotometryAmount of UV absorbed by sample is directly proportional to amount of RNAAbsorbance260nm of 1.0 = 40 g of ds RNA/mL

RNA integrityMost common rRNA molecules are 18S and 28S in eukaryotes on electrophoresis these should appear as discrete bands

Quick quantification of DNA/RNA/proteinVideo

Learning outcomes A clear understanding of the molecular structure of DNA and RNA What does 5 and 3 end mean and why is important to carefully label each end? Be able to discuss the different DNA and RNA purification methods and the problems associated with both Be able to draw flow diagrams Be able to describe how DNA and RNA are quantified once they are purified

DNA purificationFlow diagramFunction of all reagentsRNA purificationFlow diagram

DNA/RNA quantification key pointsRNA structurekey points

DNA structurekey points