Library Screening, Characterization, and Amplification

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Library Screening, Characterization, and
Amplification
•
Screening of libraries
•
Amplification of DNA (PCR)
•
Analysis of DNA (Sequencing)
•
Chemical Synthesis of DNA
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Screening of Libraries
1. Screening libraries with gene probes (DNA level):
-> Hybridisation: - Colony Hybridisation
- Plaque Hybridisation
2. Screening Expression libraries (Protein level):
-> Activity screening (-> HTS of Directed Evolution Libraries)
-> with Antibodies
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Screening of Libraries
1. Hybridisation:
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Gene Probes
-
Homologous gene probes (DNA from the same gene, same organism)
-> if you have already an incomplete clone of the gene
-> if you want to clone neighboring regulatory elements (promoters)
-> if you have cDNA clone but want the genomic clone as well
-> genetic variations between individuals (mutation causing diseases)
- Heterologous gene probe (DNA from the same gene, different organism)

-> if you have already the gene from the same gene family but different organism (insulin from
rat in order to screen human library)
- Probe generated by back translation -> degenerated oligonucleotide probe
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A degenerate oligonucleotide probe.
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Colony Hybridisation
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Plaque Hybridisation
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Screening of
Expression Libraries
with Antibodies
Primary Antibody: against protein
of interest (specific)
Secondary Antibody: against
proteins (antibodies) produced in
rabbit, mouse, bird,… (unspecific
but labeled)
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Characterization of gene products
• Restriction analysis
• Southern blot hybridisation
• PCR
• DNA sequencing
• Chromosome walking
- Characterization of large fragments -> make ordered libraries
- Identify genes (clone genes)
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Characterization of Nucleotide sequences and

protein sequences - Blots
Blots -> Transfer of target molecules to filters -> analysis of target molecules on
filters
1. Southern Blot:
-> Hybridisation of DNA (target) with DNA or RNA (Probe)
used for detection and characterization of gene fragments
2. Northern Blot:
-> Hybrisation of RNA (target) with DNA or RNA (probe)
used for detection of transcrition level (mRNA) of expressed genes (can also be done by real-
time PCR) -> analysis of gene expression
used for detection of size of transcript (length of mRNA) -> analysis of alternative splicing
3. Western Blot:
-> Interaction of Antigen with Antibody
used for detection and localization of proteins
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Detection of DNAs containing specific base
sequences by the Southern blot technique.
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Restriction fragment length polymorphism or RFLP analysis is used to identify a
change in the genetic sequence that occurs at a site where a restriction enzyme cuts.
RFLPs can be used to trace inhertitance patterns, identify specific mutations, and for
other molecular genetic techniques.
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Chromosome Walking
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PCR – Polymerase Chain Reaction
1993 Kary B Mullis received the Nobel Prize in Chemistry
1. Step -> Denaturation (94-96º C)
2. Step -> Annealing (variable Temp.)

T -> 2-4 C below melting T
3. Step -> Extension (68-72º C)
Normally around 30-35 cycles ->
around 1 mill copies
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PCR
Reaction mix:
•
Primers (15 – 30 bp) -> GC at 3’ end, not too high Tm (40-70˚C), ho hairpine
• Nucleotides (A,T, G,C)
• Buffer -> Mg
2+

•
Target DNA (around 10 ng)
• Taq Polymerase (from
Thermus aquaticus
-> thermostable)
Fidelity: -> rate of misincorporation
-> in DNA replication : 1 in 10
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nucleotides (proof reading)
-> in PCR (Taq polymerase) : 1 in 2x10
4
nucleotides
High fidelity PCR -> Pfu,… (engineered polymerases)
For Engineering purpose -> low fidelity -> introduction of mutations
- Change of salt (Mg
2+
-> Mn

2+
) and salt concentration
-
increase concentration of polymerase
- Not equal amount of nucleotides or dITP
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PCR Applications
• Amplification of DNA
•
Modification of ends for cloning (RACE)
•
Analysis of PCR products (nested primers)
• Cloning of genes (amplification from genome or library)
• Introduction of site-specific mutations
•
Joining ends (religation of different DNA molecules) without ligation
•
Invitro splicing
• Reverse Transcriptase (RT)-PCR
• Real-time PCR -> Diagnostics
•
Asymmetric PCR -> ssDNA -> sequencing
•
Detection of Infections (bacterial, viral) -> Diagnostics
• Detection of sex in prenatal cells
•
Fingerprinting -> forensic medicine
•
PCR on a Chip -> Detection of human pathogen organisms
• In situ PCR -> studying disease states, mapping chromosomes,…

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Adding of restriction sites for cloning of
a PCR product
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Joining ends without ligation
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RT-PCR – Reverse Transcriptase PCR
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Real-time PCR
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Detection of sex in
prenatal cells
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PCR fingerprinting: for identification of bacteria, forensic purpose (to assist in the
identification of individuals on the basis of their respective DNA profiles
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DNA Sequencing
1. According to Maxam- Gilbert ->
selective chemical degratation
2. According to Sanger -> polymerase
reaction with nucleotide analog
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DNA Sequencing – Sanger method
Polymerase Reaction:
5’-> 3’
-> incorporation of
ddNTP -> 3’ end has
NO OH group ->
Polymerase reaction

stops!!!