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BIOMEDICAL RESEARCH TECHNIQUES - PCR, qPCR & In Situ Hybridisation

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BIOMEDICAL RESEARCH TECHNIQUES (MEDSCI 720) ,[object Object],[object Object],[object Object],Srdjan Vlajkovic Department of Physiology
Polymerase Chain Reaction (PCR)
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
 
Polymerase Chain Reaction (PCR) ,[object Object],[object Object],[object Object]
 
PCR (Cont’d) ,[object Object],[object Object],[object Object]
PCR (Cont’d) ,[object Object],[object Object],[object Object]
 
 
 
 
Polymerase Chain Reaction
 
Multiplex PCR
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
 
 
 
Deoxyribonucleotides ,[object Object],[object Object],[object Object]
In order to use PCR, one must know the sequences which flank both ends of a given region of interest in DNA . One need not know the DNA sequence in-between.
5’ 3’ 3’ 5’ F R Primers ,[object Object],[object Object],[object Object],[object Object]
 
 
 
 
Some Applications of PCR ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object]
Real-Time qRT-PCR
Quantitation of mRNA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
 
 
Real-Time RT-PCR Chemistry ,[object Object],[object Object],[object Object],[object Object]
 
 
 
 
 
 
 
 
 
 
The principle of Taqman qPCR
Quantitation options ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object]
In situ hybridisation
In situ hybridization ,[object Object],[object Object],[object Object],[object Object]
 
Preparation of material ,[object Object],[object Object],[object Object]
Choice of probe ,[object Object],[object Object]
Probe types ,[object Object],[object Object],[object Object],[object Object]
Oligonucleotide probes ,[object Object],[object Object],[object Object],[object Object],[object Object]
Single stranded DNA probes ,[object Object],[object Object],[object Object],[object Object]
Double stranded DNA probes ,[object Object],[object Object],[object Object],[object Object]
RNA probes (cRNA probes or riboprobes) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Benefits of using oligonucleotide probes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Labeling your oligonucleotide ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
 
Probe Labelling ,[object Object],[object Object],[object Object],[object Object],Biotin Rhodamine FITC DIG-ddUTP 35 S-dATP, DIG-dUTP, Biotin-dUTP, FITC-dUTP Biotin-dATP, FITC-dATP Terminal transferase (TdT)
Detection ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object]
 
Hybridization issues ,[object Object],[object Object],[object Object],[object Object]
Permeabilization ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Pretreatment / prehybridization steps ,[object Object],[object Object],[object Object],[object Object],[object Object]
Hybridization ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Hybridization (Cont’d) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Washes ,[object Object],[object Object]
Controls ,[object Object],[object Object],[object Object]
Controls for tissue mRNA quality ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Specificity controls ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object]

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