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

PCR

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

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Mutation

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SelectionMutation

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Mutation

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SelectionMutation

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Selection

Modern Evolutionary TheoryRA Fisher, S Wright, JBS Haldane

Mutation

Recombination

SelectionMutation

Recombination

SelectionMutation

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SelectionMutation

Recombination

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Recombination

Selection

3.2 billion years

Directed Evolution

Mutation

Recombination

SelectionMutation

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Mutation

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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

PDF

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

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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