Transcript Molecular Cloning I: Restriction Enzymes, PCR, Plasmids

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
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Recombinant DNA Technology (July 22,24)
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Library
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Mutagenesis
Basic Bioinformatics
Gene Expressions (July 29,31)
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genomic library
cDNA library
Phage display library
Production of Recombinant Proteins
Reporter-fusion Proteins
Immunolocalization
Transgenic Animal
Modern Methods in Molecular Biology (August 1,14,15)
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Directed Evolution
RNAi Technology
Advanced PCR
Micro array
High Through Put (HTP) Analysis
Metagenomics (Special lecture)
Reading
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Molecular Cloning A laboratory manual
Sambrook & Russell Cold Spring Harbor
Laboratory Press c2001
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Modern Genetic Analysis
Griffiths, Anthony J.F.; Gelbart, William M.;
Miller, Jeffrey H.; Lewontin, Richard C.
New York: W. H. Freeman & Co. ; c1999
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Molecular Biology of the Cell
Alberts, Bruce; Joh nson, Alexander; Lewis,
Julian; Raff, Martin; Roberts, Keith; Walter,
Peter
New York and London: Garland Science ;
c2002
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Molecular Cell Biology
Lodish, Harvey; Berk, Arnold; Zipursky, S.
Lawrence; Matsudaira, Paul; Baltimore,
David; Darnell, James E.
New York: W. H. Freeman & Co. ; c1999
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Cell and Molecular Biology: Concepts and
Experiments, 5th Edition
Gerald Karp, Formerly of the Univ. of
Florida, Gainesville
ISBN 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
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Library
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Mutagenesis
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genomic library
cDNA library
Phage display library
Site-directed mutagenesis
Random mutagenesis
Bioinformatics
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)
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)
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
20
Lambda Phage
25
Cosmid
45
P1 phage
100
BAC
300
YAC
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
2. antibody
3. cDNA
Affinity Selection
1
Pan library with
immobilized targets
2
Wash off unbound phage
5
Pan eluted phage
3-4
3
Elute bound phage
4
Amplify overnight
© 2004 Montarop Yamabhai
6
Isolate individual colony
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 by Overlab Extension
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Bioinformatics
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What is Bioinformatics
Useful Websites
Tools
Biological Databases
Sequence Alignment
Structural Bioinformatics
Molecular Phylogenetics
Genomics/Proteomics
What is Bioinformatics
Interdisciplinary subject involving
computer and biological sciences
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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.
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The collection, organization and analysis of large amounts of biological data, using networks of computers and databases.
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The process of developing tools and processes to quantify and collect data to study biological systems logically.
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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.
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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.
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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.
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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.
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The field of science in which biology, computer science, and information technology merge into a single discipline. ...
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The field of biology specializing in developing hardware and software to store and analyze the huge amounts of data being generated by life scientists.
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information about human and other animal genes and related biological structures and processes
pharmacy.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.
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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.
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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.
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the use of computers in biological research
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use of computers in the acquisition and analysis of information relating to genes, proteins (and their structures), biological pathways and drugs
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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 genesequence dated with that about the physiological functions of the proteins whose production they simulate. ...
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the use of computers and information technology to store and analyze nucleotide and amino acid sequences and related information.
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A collective term that designates the use of computers and specialized software to analyze and retrieve data from genomic and scientific databases.
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The study of collecting, sorting, and analyzing DNA and protein sequence information using computers and statistical techniques.
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The science of managing and analyzing biological data using advanced computing techniques.
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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 Websites
USA
• NCBI
http://www.ncbi.nlm.nih.gov/
Europe
• EBI
http://www.ebi.ac.uk/
Google search
www.google.com
Tools
• Bioinformatic Software
– $$$ buy from company
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Vector NTI
Mac Vector
Sequencer
Bio edit (free software)
– Free web-based software
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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
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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
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Genome mapping
– Low resolution : using genetic markers
– Highest resolution : complete sequence of the whole genome
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Genome sequencing; assembly
– Full shotgun
– Hierarchical approach
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Genome annotation
– Gene finding, naming
– Assigning function to the gene
– The exact number of genes in human genomes is not known
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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
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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
promoter
RBS….ATG
RBS….ATG
Tag
MCS
MCS
Tag
stop
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 Cloning
NT Nuclear Transfer
Modern Methods
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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 Theory
RA Fisher, S Wright, JBS Haldane
Recombination
Mutation
Selection
Modern Evolutionary Theory
RA Fisher, S Wright, JBS Haldane
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Selection
Selection
Selection
Selection
Selection
Selection
Modern Evolutionary Theory
RA Fisher, S Wright, JBS Haldane
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Selection
Selection
Selection
Selection
Selection
Directed Evolution
3.2 months/yrs
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Recombination
Recombination
Recombination
Recombination
Recombination
Selection
Selection
Selection
Selection
Selection
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Selection
Selection
Selection
Selection
Selection
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 doublestranded 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
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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
Ct
Cycle threshold
movie
• Real-time PCR Animation - PCR and Realtime 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
Example
a 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)
template-independent addition of a single A at the 3′-end of PCR products by some thermostable DNA polymerases.
Deep Vent®
Pfu
Pwo
>95% Blunt
>95% Blunt
Blunt
N.A.
Varies
Yes
No
No
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Taq
Tfl
Tth
Resulting DNA
ends
3′-A
3′-A
3′-A
5′->3′
Yes
Yes
No
No
Tli
(Vent®)
"Long PCR"
enzyme
mixes†
exonucle
ase
activity
3′->5′
exonucle
ase
activity
NESTED PCR
A 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
55 45- 55
55 45
DNA Microarray
High Throughput Analysis
HTP
• 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