PCR, RT-PCR, FISH

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]

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]

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]

Quantitation options ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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]

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]

PCR, RT-PCR, FISH

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PCR, RT-PCR, FISH