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304512 Molecular Biology and Recombinant DNA
Technology
Montarop Yamabhai
Schedule: Tue,Th, Fri 10 am- 1 pm, 9-12
July• Tue 22 B1205 (11-13)
• Thr 24 B1114 (11-13)
• Tue 29 B1205 (11-13)
• Thr 31 B1205 (11-13)
August• Fri 1 B1122 (9-12), B1123 (10-12)
• Thr 14 B1114 (11-13)
• Fri 15 B1122 (9-12), B1123 (10-12) [Special lecture]
Topic• Recombinant DNA Technology (July 22,24)
• Library– genomic library– cDNA library– Phage display library
• Mutagenesis• Basic Bioinformatics
• Gene Expressions (July 29,31)• Production of Recombinant Proteins• Reporter-fusion Proteins • Immunolocalization• Transgenic Animal
• Modern Methods in Molecular Biology (August 1,14,15)• Directed Evolution• RNAi Technology• Advanced PCR• Micro array• High Through Put (HTP) Analysis• Metagenomics (Special lecture)
Reading• Molecular Cloning A laboratory manual
Sambrook & Russell Cold Spring Harbor Laboratory Press c2001
• Modern Genetic Analysis Griffiths, Anthony J.F.; Gelbart, William M.; Miller, Jeffrey H.; Lewontin, Richard C.New York: W. H. Freeman & Co. ; c1999
• Molecular Biology of the Cell Alberts, Bruce; Joh nson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, PeterNew York and London: Garland Science ; c2002
• Molecular Cell Biology Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E.New York: W. H. Freeman & Co. ; c1999
• Cell and Molecular Biology: Concepts and Experiments, 5th Edition Gerald Karp, Formerly of the Univ. of Florida, GainesvilleISBN 978-0-470-04217-5, ©2008, 864 pages
Evaluation
• Assignments 40%
Select a company that sale any “advanced” biotech products that you like. Then, write a “product review” describing this product in as much detail as possible but no more than one A4 page
• Exam 60%
Opeb book, 3 hrs. ? 22 August 2008?
Advanced Recombinant DNA Technology
• Library– genomic library– cDNA library– Phage display library
• Mutagenesis– Site-directed mutagenesis– Random mutagenesis
• Bioinformatics
Chapter 18: Techniques in Cell and Molecular Biology
Mechanism of Recombination Video (size: 360 x 240 or 588 x 392)
Polymerase Chain Reaction Video (size: 360 x 240 or 588 x 392)
Chapter 10: The Nature of the Gene and the Genome
Chargaff's Ratios Video (size: 360 x 240 or 588 x 392)
Base Pairing Video (size: 360 x 240 or 588 x 392)
Public Project Sequencing Video (size: 360 x 240 or 588 x 392)
Chapter 11: Expression of Genetic Material: From Transcription to Translation
Transcription Video (size: 360 x 240 or 588 x 392)
Triplet Code Video (size: 360 x 240 or 588 x 392)
Translation Video (size: 360 x 240 or 588 x 392)
Chapter 12: The Cell Nucleus and the Control of Gene Expression
How Much DNA Codes for Protein Video (size: 360 x 240 or 588 x 392)
How DNA is Packaged Video (size: 360 x 240 or 588 x 392)
Microarray Video (size: 360 x 240 or 588 x 392)
Chapter 13: DNA Replication and Repair
Replicating the Helix Video (size: 360 x 240 or 588 x 392)
Mechanism of Replication Video (size: 360 x 240 or 588 x 392)
Chapter 16: Cancer
Microarray Video (size: 360 x 240 or 588 x 392)
Tumor Growth Video (size: 360 x 240 or 588 x 392)
Review
Library
• A collection of DNA in different forms– Genomic Library– cDNA Library– Phage Display Library
• Micro organisms used– Bacteria– Bacteriophage (Phage)
Types of Cloning Vector
Types size of cloned DNA (kb)Plasmid 20Lambda Phage 25Cosmid 45P1 phage 100BAC 300YAC 1000
Bacterial Library
Phage Library
Lambda λ Phage
Filamentous Phage, M13, Fd
Genomic Library
mRNA
cDNA Library
Select for ampicilin resistance colonies
Screening of the Library
• DNA hybridization• PCR• Functional Screening
Membrane hybridization assay
Functional Screening
Phage Display Library
What can be displayed
............1. Random peptide (X)12
3. cDNA
2. antibody
1Pan library withimmobilized targets
2Wash off unbound phage
3Elute bound phage
4Amplify overnight
5Pan eluted phage
6Isolate individual colony
3-4
© 2004 Montarop Yamabhai
Affinity Selection
Phage ELISA
HRP
Mutagenesis
• Purpose– Study regulatory regions of the genes– Study structure-function relationship
of protein– Alter activity of enzymes or proteins
• Types– Random Mutagenesis– Site-directed Mutagenesis
Random Mutagenesis
• Chemical treatment• Error-prone PCR• DNA shuffling
Error Prone PCR
• Non-proofreading polymerase, i.e. Taq
• Low annealing temperature• Low/unequal dNTP concentration• High Mg2+
• High cycle number 40-80• Incorporation of Mn2+ ion (0.5-.5
mM)
DNA Shuffling
Site-Directed Mutagenesis
• Kits from various company• PCR-based mutagenesis
Site-directed Mutagenesis by PCR Site specific mutagenesis byOverlab Extension
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Bioinformatics• What is Bioinformatics• Useful Websites• Tools• Biological Databases• Sequence Alignment• Structural Bioinformatics• Molecular Phylogenetics• Genomics/Proteomics
What is Bioinformatics
Interdisciplinary subject involvingcomputer and biological sciences
• the science of informatics as applied to biological research. Informatics is the management and analysis of data using advanced computing techniques. Bioinformatics is particularly important as an adjunct to genomics research, because of the large amount of complex data this research generates.www.food.gov.uk/science/ouradvisors/toxicity/cotmeets/49737/49750/49831
• The collection, organization and analysis of large amounts of biological data, using networks of computers and databases.www.abc.net.au/science/slab/genome2001/glossary.htm
• The process of developing tools and processes to quantify and collect data to study biological systems logically.www.als.net/als101/glossary.asp
• the assembly of data from genomic analysis into accessible forms. It involves the application of information technology to analyze and manage large data sets resulting from gene sequencing or related techniques.www.doylefoundation.org/icsu/glossary.htm
• the use of computers in solving information problems in the life sciences. It mainly involves the creation of extensive electronic databases on genomes, protein sequences etc. Also involves techniques such as three-dimensional modelling of biomolecules and biological systems.www.universityscience.ie/pages/glossary.php
• The use of computers to handle biological information. The term is often used to describe computational molecular biology – the use of computers to store, search and characterize the genetic code of genes, the proteins linked to each gene and their associated functions.www.syngenta.com/en/about_syngenta/research_tech_gloss.aspx
• A broad term to describe applications of computer technology and information science to organize, interpret, and predict biological structure and function. Bioinformatics is ususally applied in the context of analyzing DNA sequence data. Biomagnification: a problem associated with the introduction of xenobiotic compounds into the biosphere in which the concentration of the compound increases as it passes up the food chain.www.plpa.agri.umn.edu/scag1500/definitions.html
• The field of science in which biology, computer science, and information technology merge into a single discipline. ...www.epa.gov/comptox/glossary.html
• The field of biology specializing in developing hardware and software to store and analyze the huge amounts of data being generated by life scientists.www.nigms.nih.gov/news/science_ed/genetics/glossary.html
• information about human and other animal genes and related biological structures and processespharmacy.ucsf.edu/glossary/i/
• is research, development or application of mathematical tools and approaches for expanding the use of biological, medical, behavioral or health data. This includes methods to acquire, store, organize, archive, analyze or visualize data.www.mayouminnesotapartnership.org/glossary.html
• The use of computers to collect, analyze and store genomics information.pbi-ibp.nrc-cnrc.gc.ca/en/media/glossary.htm
• The use of computers, laboratory robots and software to create, manage and interpret massive sets of complex biological data.www.med.umich.edu/genetics/glossary/
• The collection and storage of information about genomics in databases.www.pub.ac.za/resources/glossary.html
• The management and analysis of data from biological research.www.biotech.ca/EN/glossary.html
• Description: A scientific discipline that comprises all aspects of the gathering, storing, handling, analysing, interpreting and spreading of biological information. Involves powerful computers and innovative programmes which handle vast amounts of coding information on genes and proteins from genomics programmes. ...europa.eu.int/comm/research/biosociety/library/glossarylist_en.cfm
• The discipline of obtaining information about genomic or protein sequence data. This may involve similarity searches of databases, comparing your unidentified sequence to the sequences in a database, or making predictions about the sequence based on current knowledge of similar sequences. Databases are frequently made publically available through the Internet, or locally at your institution.bioinfo.cnio.es/docus/courses/SEK2003Filogenias/seq_analysis/Glossary.html
• An interdisciplinary area at the intersection of biological, computer, and information sciences necessary to manage, process, and understand large amounts of data, for instance from the sequencing of the human genome, or from large databases containing information about plants and animals for use in discovering and developing new drugs.www.isye.gatech.edu/~tg/publications/ecology/eolss/node2.html
• the use of computers in biological researchwww.epidauros.com/cms/en/pharmacogenetics/glossary.html
• use of computers in the acquisition and analysis of information relating to genes, proteins (and their structures), biological pathways and drugswww.serenex.com/Page87
• the organisation and use of information on biological and molecular subjects. This includes organising biomolecular databases, managing the quality of data input, getting useful information out of such databases, and integrating information from disparate sources. One application of bioinformatics is to bring together gene-sequence dated with that about the physiological functions of the proteins whose production they simulate. ...www.biotechnology.vic.gov.au/info/glossary.asp
• the use of computers and information technology to store and analyze nucleotide and amino acid sequences and related information.www.oup.com/uk/booksites/content/0199264724/student/glossary.htm
• A collective term that designates the use of computers and specialized software to analyze and retrieve data from genomic and scientific databases.www.painceptor.com/page.asp
• The study of collecting, sorting, and analyzing DNA and protein sequence information using computers and statistical techniques.www.bscs.org/onco/glossary.htm
• The science of managing and analyzing biological data using advanced computing techniques.www.patientshelpingdoctors.org/phd/definitions
• Bioinformatics or computational biology is the use of techniques from applied mathematics, informatics, statistics, and computer science to solve biological problems. Research in computational biology often overlaps with systems biology. Major research efforts in the field include sequence alignment, gene finding, genome assembly, protein structure alignment, protein structure prediction, prediction of gene expression and protein-protein interactions, and the modeling of evolution. ...en.wikipedia.org/wiki/Bioinformatics
Useful Server WebsitesUSA• NCBI http://www.ncbi.nlm.nih.gov/
Europe• EBI http://www.ebi.ac.uk/
Google searchwww.google.com
Tools
• Bioinformatic Software– $$$ buy from company
• Vector NTI• Mac Vector• Sequencer• Bio edit (free software)
– Free web-based software• DNA sequence analysis• Primer design• DNA translation tools• Structure prediction• Restriction site analysis• Sequence alignments• etc
Biological Databases
• Primary Database• Secondary Database• Specialized Database
Primary Database
• Raw nucleic acid sequence– Genbank– EMBL– DDBJ
• Use different format to present data• These databases are closely connected and
exchanged data daily
• 3D structure : PDB• Protein and nucleic acid structure• Atomic coordinates from X-ray and NMR
Secondary Database
• Computational processed information
• Provide sequence annotation• SWISS-PROT• TrEMBL (translated nucleic acid
sequence from EMBL)• Other : UniPort, Pfam, Blocks, DALI
Specialized Database
• Focus on particular organisms– Flybase– Wormbase– AceDB, TAIR
• Focus on functional analysis– Genbank EST– Microarray gene expression database
Important• Many database are connected
– NCBI are most integrated• Reliability !• There are many errors in the database
– Sequencing error (especially before 1990s)
– Redundancy• Non-redundant database• UniGene (coalesce EST)
Information Retrieval
• Use Boolean operation = join a series of keywords
• Text-based search• Provide access to multiple database
for retrieval of integrated search result
• Entrez (NCBI)• SRS (Seq retrival system from EBI)
Sequence Alignment
• Heart of bioinformatic analysis• A consequene of evolution• Help to identify evolution
relationship
Sequence homology ≠ Sequence similarity
• Sequence homology is a “quantitative term showing common evolution origin
• Sequence similarity is a “quantitative term” calculating from sequence alignment (% similarity)
• From % similarity one can conclude that the sequence is homolog or non-homolog
Sequence similarity & Sequence identity
• Same for DNA• Different for protein
– Protein similarity means % of similar physicochemical characteristic
– Protein identity means % of match of the same amino acid sequence
• Formula (%)– Ls(i) x 2 / La+Lb x 100– Ls(i) / La x100
• La is the length of shorter sequence
Similarity Searching
• Submit “query sequence” to perform pairwise comparison using computational process
• Use heuristic method• BLAST
– Developed in 1990s– Variations: BLASTN, BLASTP, BLASTX,
TBLASTN, TBLASTX, NBLAST
• FASTA• Significant determined from E-value
Important
• Protein sequence is better (more sensitive)
• Not guarantee to find all homolog• Must be followed by independent
alignment programs• Must filter out LCRs (low complexity
regions eg. Repetitive sequence)
Multiple Sequence Alignment
• Reveal more information than pair wise alignment
• Identify conserved or critical amino acid
• Required for phylogenic analysis and prediction of protein structure
• For designing degenerated primers for PCR-cloning
Methods / Program• Dynamic program• Heuristic approach
– Clustal; ClustalW, ClustalX– T-Coffee– Poa– PRALINE– PRRN, etc
• Editing– BioEdit– Rascal
Important
• No alignment program is perfect• Combine results from multiple
program• The alignment should be refine
manually• Protein sequence alignment is more
accurate and should be aligned first
Prediction of Gene and Promoter
• Very difficult• Prokarotic genomes are much
easier to predict• The good program is being
developed– GeneMark– Glimmer
Molecular Phylogenetic
Evolution
• Development of biological form from preexisting form through natural selection and mutation
• Protein or DNA sequence are molecular fossils
Nature 392:917-920, 1998
Major Assumptions
• Molecular sequences used in phylogenetic construction are homologous
• Evolutionary tree is always binary• Each position in a sequence evolve
independently
Types of Phylogenetic trees
Steps
• Choosing molecular markers• Performing multiple sequence
alignment• Choosing a model of evolution• Determining a tree building
method• Assessing tree reliability
Selection of molecular markers
• For closely related organisms (individual within populations), DNA sequence is used, as it evolves fast
• For more widely divergent group (different species of bacteria, fungi) use slowly evolving sequence such as ribosomal RNA or protein
• For greatly different organisms (bacteria and eukaryote) use conserve protein sequence– DNA sequence can be biased– Protein sequence allow more sensitive alignment
Important
• Phylogenetic tree construction is a complicated process
• None of the methods are guarantee to find a correct tree
• At least 2 methods should be used for any phylogenetic analysis
Structural Bioinformatics
• Protein functions are determined by their structures
• Essential elements in bioinformatics• 20 amino acids are building blocks of
protein• Amino acids are linked by peptide bond• Conformation (folding) of protein is
determined by dihedral angle (phi and psi)
Ramachandran Plot
3D structure of protein
Can be determined by• X-ray crystallography
– Protein need to be grown into large crystal; bottle neck– The x-ray are relected by electron cloud surrounding the
atoms, diffraction patterns are converted into electron density map
– 2 methods are used to resolved the structures• Molecular replacement• Multiple isomorphous replacement
– R factor is used to determined the quality of the model, ranging from 0.0 – 0.59
• NMR (nuclear magnetic resonance)– Detect spinning pattern of atomic nuclei in magnetic field– Protein are in solution, so it is mobile and vibrating, thus a
number of different models will be constructed– Limit to <200 amino acid residues, use radioisotope
Protein visualization
• PDB only contains x, y, z coordinate of atoms
• Widely used and freely available software– RasMol, RasTop– Swiss-PDBViewer– WebMol, Chime, Cn3D
• Unix software– Molscript– Ribbons– Grasp
Other software
• Software for structure comparison– DALI
• Software for protein classification– SCOP– CATH
3D Structure Prediction
• Theoretical alternative to experimental approaches
• There are 3 computational approaches– Homology modeling : most accurate
• Divided into 6 steps : 1) template recognition, 2) sequence recognition, 3) backbone generation, 4) loop building, 5) side chain building, 6) model refinement and evaluation
– Threading or fold recognition– Ab initio recognition
• Comprehensive modeling program– Modeller, Swiss-Model, 3D-JIGSAW
Genomics and Proteomics
• Genomics is the study of genome involving simultaneous analysis of a large number of genes, using automated high-throughput machine
• Genomic study can be divided into 2 parts– Structural genomics– Functional genomics
Structural Genomics• Genome mapping
– Low resolution : using genetic markers– Highest resolution : complete sequence of the whole genome
• Genome sequencing; assembly– Full shotgun– Hierarchical approach
• Genome annotation– Gene finding, naming– Assigning function to the gene– The exact number of genes in human genomes is not known
• Comparative genomics– Help to discover potential operon and assign putative funciton– Conserved gene order among prokaryotes often indicate protein
physical interaction (e.g. protein in the same metabolic pathway)– BLASTZ or LAGAN are the two best programs for genome
comparison
Level of Analysis
• Cytological map– Pattern on the chromosome
• Genetic map– Genetic marker
• Physical map (Restriction Map)• DNA sequence
Sequencing Approaches
• Shot gun sequencing approach– Genomic DNA many short fragments
cloned sequencing each clone assemble sequence by aligning and removing overlaps (need high capacity software)
• Hierarchical sequencing approach– Genomic DNA long fragments cloned
into BAC library with completed physical map subclone library from each BAC clone sequencing each subclone assemble sequence by aligning and removing overlaps
Functional Genomics
• Study gene function at the whole genome level using high throughput approach
• Simultaneous analysis of all genes in a genome
• Transcriptome : all expressed genes• 2 approaches for analysis
• Sequence-based (ESTs)• Miroarray-based : most popular method to study
gene expression
Proteomics
• An entire set of expressed proteins in a cell
• Simultaneous study of all the translated proteins in a cell
• High-throughput analysis of the protiens– Protein expression– Posttranslational modification– Protein sorting– Protein-protein interaction
Traditional proteomic analysis
Gene Expression• Production of Recombinant
Proteins• Reporter-fusion Proteins• Immunolocalization• Transgenic Organisms• cDNA library• Northern / Western Blot
Analysis• Micro array
Detection of Proteins
Gene Expression
• Proteins can be made in large amount in various organisms through the use of expression vector
• Expression of proteins is used to– Production of large amount of proteins– Study the biological function of different
proteins
• Various organisms (systems) can be used to expressed foreign proteins
Gene Expression Systems• Bacteria expression systems
– E.coli– Bacillus subtilis
• Yeast expression system– S.cerevisiae– Pichia pastoris
• Mammalian expression system– Primary cells or cell lines (human, mouse)– Transgenic animals
• Insect expression system• Plant expression system
Production of Recombinant Proteins
• E. coli expression system• Other bacterial expression
systems• Eukaryotic expression
systems
Expression Vector
promoter RBS….ATG MCSTag stop
promoter RBS….ATG MCS Tag stop
inducer
Bacterial Gene Expression
Lac Operon
Artificial inducer
• Isopropyl-β-D-thio-galactoside (IPTG)
Eukaryotic Gene Expression
Putting genes into cells
• Tranformation - Bacteria, Yeast• Transfection - metazoa
– Chemical– Electrical– Viral infection
Expression of Fusion Proteins
• Tagged-protein for Purification– GST– 6xHis, myc epitope, Flag
• Reporter Proteins– Green Fluorescence proteins (GFP)– LacZ
Transgenic animal
• Transgenic mice• Knock out mice• Animal cloning
http://gslc.genetics.utah.edu/features/knockout/index.cfm
Animal CloningNT Nuclear Transfer
Modern Methods
• Directed Evolution• RNAi Technology• Real time PCR• Micro array• High Through Put (HTP) Analysis• Metagenomics (Special lecture)
Directed Evolution
•Principle of directed evolution techniques
•Applications of directed evolution techniques
Modern Evolutionary TheoryRA Fisher, S Wright, JBS Haldane
Recombination
Mutation Selection
Modern Evolutionary TheoryRA Fisher, S Wright, JBS Haldane
Mutation
Recombination
SelectionMutation
Recombination
Mutation
Recombination
SelectionMutation
Recombination
Mutation
Recombination
SelectionMutation
Recombination
Mutation
Recombination
SelectionMutation
Recombination
Mutation
Recombination
SelectionMutation
Recombination
Mutation
Recombination
Selection
Modern Evolutionary TheoryRA Fisher, S Wright, JBS Haldane
Mutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
Selection
3.2 billion years
Directed Evolution
Mutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
Selection
Mutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
Selection
Mutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
Selection
Mutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
SelectionMutation
Recombination
Selection
3.2 months/yrs
Directed Evolution
RNAi• RNA interference • A mechanism for RNA-guided
regulation of gene expression in which double stranded RNA inhibits the expression of genes with complementary nucleotide sequences
• Powerful tool in molecular biology and have many potentials application in medicine and biotechnology
History• First observed in plants but
don’t know why?
• Craig C. Mello and Andrew Fire's 1998 Nature paper reported a potent gene silencing effect after injecting double stranded RNA into C. elegans.[5] They observed that neither mRNA nor antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. This work represented the first identification of the causative agent of a previously inexplicable phenomenon. They were awarded the Nobel Prize in Physiology or Medicine in 2006 for their work.
The most interesting aspects of RNAi are the following
• dsRNA, rather than single-stranded antisense RNA, is the interfering agent
• it is highly specific • it is remarkably potent (only a few dsRNA
molecules per cell are required for effective interference)
• the interfering activity (and presumably the dsRNA) can cause interference in cells and tissues far removed from the site of introduction
RNAi focus from Nature Reviews: RNA interference – Animations
Advanced PCR
• Real time PCR• PCR cloning• Nested PCR• Touch down PCR
Real Time - PCR
• RT- PCR not Reverse Transcription PCR• Both quantitative and qualitative• Two types of detection
– Double stranded DNA dyes• such as SYBR Green
– Fluorescent reporter probe • More specific• More expensive
CtCycle threshold
movie
• Real-time PCR Animation - PCR and Real-time PCR principles and comparison
• Real Time PCR Tutorial by Dr Margaret Hunt, University of South Carolina, September 5, 2006
• Real-Time PCR Vs. Traditional PCR tutorial from Applied Biosystems (link opens a PDF document)
PCR Cloning
• Primer Design– Specific primers– Degenerated primers– Nested primers
• Amplification– High-fidelity DNA polymerase– Hot start– Touch down PCR
• Clone into appropriate vector– Compatible restriction sites– Poly T (pGEM T easy)– No ligation (Topo cloning)
Degenerated Primers
Examplea protein motif:
W D T A G Q E Trp Asp Thr Ala Gly Gln Glu 5' TGG GAY ACN GCN GGN CAR GAR 3'
where the Y = C or T, R = G or A, N = G, A, T or C.
(This gives a mix of 256 different oligonucleotides.)
Align Design
pGEM-T (Promega)
Taq Tfl Tth Tli (Vent®)
Deep Vent® Pfu Pwo "Long PCR" enzyme mixes†
Resulting DNA ends
3′-A 3′-A 3′-A >95% Blunt >95% Blunt Blunt N.A. Varies
5′->3′ exonuclease activity
Yes Yes Yes No No No No Yes
3′->5′ exonuclease activity
No No No Yes Yes Yes Yes Yes
template-independent addition of a single A at the 3′-end of PCR products by some thermostable DNA polymerases.
NESTED PCRA powerful method to amplify specific sequences of DNA from a
large COMPLEX mixture of DNA.
Touch Down / Step down one-step procedure for optimizing PCRs
45-55
55-45
55
DNA Microarray
High Throughput AnalysisHTP
• The study of DNA or protein in a large scale (103-1010) molecules at the same time
• Genomic – DNA microarray• Proteomic
– Protein/Antibody array– Protein analysis by mass spectrometry– 2D gel analysis
• Need robot• For drug discovery
No Ligation / TOPO Cloning
The study of protein-protein interactions
• Yeast two-hybrid system• Phage display technology• Pull-down experiments