Library Screening, Characterization, and Amplification

Library Screening, Characterization, and Amplification • Screening of libraries • Amplification of DNA PCR • Analysis of DNA Sequencing • Chemical Synthesis of DNA... Screening libraries

Library Screening, Characterization, and

Amplification

• Screening of libraries

• Amplification of DNA (PCR)

• Analysis of DNA (Sequencing)

• Chemical Synthesis of DNA

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

Screening of Libraries

1 Hybridisation:

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)

A degenerate oligonucleotide probe.

Colony Hybridisation

Plaque Hybridisation

Secondary Antibody: against

proteins (antibodies) produced in

rabbit, mouse, bird,… (unspecific

but labeled)

Characterization of gene products

- Characterization of large fragments -> make ordered libraries

- Identify genes (clone genes)

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 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

Detection of DNAs containing specific base sequences by the Southern blot technique.

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

Chromosome Walking

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)

• Target DNA (around 10 ng)

Fidelity: -> rate of misincorporation

High fidelity PCR -> Pfu,… (engineered polymerases)

For Engineering purpose -> low fidelity -> introduction of mutations

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,…

Adding of restriction sites for cloning of

a PCR product

Joining ends without ligation

RT-PCR – Reverse Transcriptase PCR

Real-time PCR

Detection of sex in

prenatal cells

DNA Sequencing

1 According to Maxam- Gilbert ->

selective chemical degratation

2 According to Sanger -> polymerase

reaction with nucleotide analog

DNA Sequencing – Sanger method

Cycle Sequencing - PCR

DNA sequencing by primer walking

Chemical synthesis of DNA