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Molecular Biology Techniques. Nicky Mulder Acknowledgements: Anna Kramvis for lecture material (adapted here). Experiments for different cell processes. Two levels of experiment. Small-scale -1-10 genes/proteins: PCR Restriction enzymes Cloning Hybridization - PowerPoint PPT Presentation
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Molecular Biology Techniques
Nicky Mulder
Acknowledgements: Anna Kramvis for lecture material (adapted here)
Experiments for different cell processes
Two levels of experiment
Small-scale -1-10 genes/proteins:PCR Restriction enzymesCloningHybridization
Large-scale 100-10000 genes or whole genome -> High-throughput biology
Polymerase Chain Reaction
Fnlmh.ufl.edu/cowries/PCR
Agarose gel electrophoresis
Agarose is used to form a gel Gel is placed in solution with an anode and
cathode DNA has net negative charge from sugar-
phosphate backbone –migrates towards anode Migration speed is determined by size Run DNA with some markers of known size Visualized by ethidium bromide –flouresces in
UV light
Results on a gel
Restriction enzymes recognize specific or defined 4 to 8 base pair sequences on DNA and cut
Microorganism Enzyme Sequences Notes
Haemophilus aegitius
HaeIII 5’…GG CC..3’3’…CC GG..5’
Blunt end
Haemophilus haemolytica
HhaI 5’…GC G C..3’3’…CG C G..5’
3’ single strand
Escherichia coli EcoRI 5’…G AATT C..3’3’…C TTAA G..5’
5’ single strand
Restriction enzymes
Restriction maps
Restriction maps on gel
www.cbs.dtu.dk
+
-
Use of restriction enzymes
Cloning
Restriction fragment length polymorphism
Restriction Fragment Length Polymorphism (RFLP)
M1M1 M2M2 A2A2 A1A1 DD
StuStuII
1.5 kb1.5 kb
1.0 kb1.0 kb
750 bp750 bp
500 bp500 bp
400 bp400 bp
300 bp300 bp
200 bp200 bp
145 bp145 bp
448 bp448 bp
593 bp593 bp671 bp671 bp
uncutuncut
concon
CloningCloning
www.biodavidson.edu
Cloning vectors
Features:
Antibiotic resistance gene
Another marker gene (lacZ*)
Specific promoter
Multiple cloning site
*Lac Z gene, encodes beta-galactosidase- causes bacteria expressing the gene to appear blue when grown on a medium that containing X-gal
Other vectors
Bacterial artificial chromosomes Yeast artificial chromosomes Organism-specific vectors Expression vectors
Prokaryote gene transfer
Conjugation –transfer between bacteria by direct contact
Transduction –transfer of DNA via a virus Transformation –uptake of DNA from
environment by competent cells
Southern Hybridization
http://www.cdc.gov/ncidod/eid/vol6no1/images/vanderpoel1b.gif
Northern Hybridization
http://www.molecularstation.com/images/northern-blot-med.jpg
Western Hybridization
http://www.steve.gb.com/images/science/western_blotting.png
High-throughput biology
Move away from single gene focus and bottom-up approach
Studying multiple genes at once Using new technologies Moving from genotype to phenotype Trying to find function of sets of genes:
Functional genomics
Functional genomics experiments
DNA sequencing and analysis Mutagenesis and gene disruption DNA microarrays (transcriptomics) Proteomics (protein expression, 2D
gels, protein-protein interactions) Structural genomics Metabolomics
Functional genomics & Bioinformatics
Large-scale experiments generating vast amounts of data
Data needs sorting and analysis Bioinformatics allows:
Tracking of samplesAutomating data captureData storage and analysisData mining to convert data into biological
research
DNA sequencing technologies
Sanger sequencing method (chain termination) Dideoxynucleotide triphosphates (ddG/A/T/C/TP, lack
3-OH), labelled primers and DNA polymerase -4 reactions –run on gel
Dye terminator sequencing Label terminators with diff dyes –single reaction, use
capillary electrophoresis
High-throughput sequencing Parallel reactions, DNA on surfaces –sequencing by
synthesis and detection of fluorescence
www.bio.davidson.edu/Courses/Bio111/dnaseq2.gif
Sanger sequencing method
Automated sequencing
Automated sequencing
Genome sequencing
To sequence a fragment of DNA: subclone fragment into vector- plasmid (2kb),
cosmid (40kb), BAC (>100kb) or YAC (1Mb)Grow cells and purify DNASequence user flourescent dye labels and
laser detection –can get 300-800bp per read Problem is if fragment is too big –not
covered by reads
Whole genome shotgun
Need to fragment the DNA, sequence the pieces and then assemble them
Need to over-sample to get good overlaps May still get gaps using this approach, but can
design new primers for additional sequencing Repeats are an issue –can cause incorrect
assembly Shotgun sequencing works for small genomes
like bacterial genomes
Sequencing complex genomes
As the complexity increases so does likelihood of incorrect assembly –eukaryotes has many repeats
Genome maps are important and form a guide for showing positions of genes and features
Eukaryotic genomes are fragmented into 1.5Mb bits and cloned into BACs, then a shotgun approach is used for each BAC –hierarchical shotgun sequencing
These “contigs” are assembled as before, and mapped onto genome using markers (genetic map)
Hierarchical shotgun sequencing
International Human Genome Sequencing Consortium, 2001, Nature 409, pg 860-921.
Assembly
PHRAP Assembly: Align fragments, consensus quality
CONSED sequence
editing
PHRED Base calling, trace
quality, Crossmatch –finds vector
Highest quality reads used for
consensus
Hierarchical shotgun
sequencing
Genome Annotation
Two main levels: Structural Annotation – Finding genes and other
biologically relevant sites thus building up a model of genome as objects with specific locations
Functional annotation – Objects are used in database searches (and expts) aim is attributing biologically relevant information to whole sequence and individual objects
Genome structure
Genes, pseudogenes, introns, exons, intergenic regions
Proteins
Functional annotation
Gene prediction Promoter prediction
Translation BLAST Signatures 2D structure 3D structure
Annotation can be at different levels
Function, structure
Gene regulation
Cellular process, localisation
Interactions, pathways
Gene expression -Transcriptomics
Microarrays ChIP on chip (Chromatin IP on
microarrays)
Slide with target deposited
label cDNA (probe)
hybridise labelled probe to slide
wash slides
scan
analyse results
Microarray overview
Microarray data analysis
Experimental design
Normalization
Pre-processing
Data analysis
Data mining
Image processing
Co-regulated genes have correlated expression patterns
Data mining
-AB02387 -SB07593 -AA00498 -AC008742 -AB083121
Add gene identifiers
-RNA polymerase -Glycosyl hydrolase -Phosphofructokinase -Transcription factor -Glucose transporter
Add gene descriptions
-GO0003456 -GO0006783 -GO0142291 -GO0054198 -GO0000234
Add GO terms
Map onto pathways
Proteomics Large-scale study of proteins to determine
their function Proteome is protein complement of the
genome Includes the study of:
Protein structure and functionProtein-protein interactions Protein expressionProtein localizationProtein modifications
Proteomics studies
Mass spectrometry
Xray, NMR
Mass spectrometry
Localization studies
Workflow of a proteomics experiment
Sample can be from patient cohort, cell selection, fraction, etc.
Sample preparation
Protein separation
Protein selection
Protein identification
Different separation techniques, e.g. 2-D PAGE, HPLC, ICAT, etc.
Depends on separation method
Usually mass spectrometry
Protein separation
2D PAGE Gel-free
systems: ICAT HPLC
Mass spec –digest proteins further
Protein separation -2D PAGE
pH gradient
Siz
e gr
adie
nt
Bioinformatics component
Sample tracking Image capture Image analysis and comparison:
Measuring intensitiesRemoving background noiseFinding difference between gels
After 2D PAGE
Mass spectometry Digest proteins with e.g. trypsin (lysine or
arginine) Proteins ionized and brought into gas phase Move through mass analyzer which separates
them based on mass Detector records presence of ions
Protein identification (MS)Peptide Fragment Fingerprinting (PFF)
MS/MS or Tandem MS
Peptide identification (MS/MS)
VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHK
VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHK
denaturedigest with trypsin
V HLTPEEKVH LTPEEKVHL TPEEKVHLT PEEKVHLTP EEKVHLTPE EKVHLTPEE K
Mass spec
mass spectrum
compare with theoretical peptide spectra;ID = best similarity
Recognises lysine (K) & arginine (R)
Summary Aim of molecular biology experiments is to
understand biology Find gene/protein functions See what is causing a phenotype see if/when a gene or protein is expressed
Cloning exercise