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