DNA genotyping lab

Summary
In this laboratory session, you will use PCR to detect genetic alterations in DNA samples. You will gain the specific skills of DNA extraction, qPCR set up and qPCR data analysis. You need to be able to pipette low volumes correctly; if you are unsure about your technique ask the lab demonstrator or review the pipetting instructions at the end of this document.

You will extract DNA from a series of patient tumour cell samples (1 - 4). You will use these patient DNA samples to detect a Single Nucleotide Polymorphism using Taqman-based technology. In addition, you will use Real-Time PCR-based mutation detection methods.

The some of the tumour samples may contain the V600E mutation in the B-RAF gene. The qPCR SNP analysis will identify which samples contain this mutation compared to control DNA with known mutation status

The likely order of procedures is as follows:

1) Extract DNA for Taqman mutation/SNP detection.
2) Run Taqman mutation detection using Real-Time PCR.
3) Perform basic bioinformatics in the afternoon.

Data analysis, dry lab and bioinformatics tools
In the afternoon dry lab (computer room) you will gain bioinformatic skills associated with handling and analysing DNA sequences from online databases (NCBI: BLAST tools). During these sessions, you will be given the skills to solve some mutation detection-related problems as set out in the lab book.

DNA extraction: (Taken from QiaAmp protocol)
***Ensure that the Ethanol has already been added to the buffers prior to starting.

1) Re-suspend patient tumour cells in 200 µl PBS.

2) Add 20 µl of proteinase K.

3) Add 200 µl Buffer AL to the sample. Mix by pulse-vortexing for 15 seconds.

4) Incubate at 56 °C for 10 minutes.

5) Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid. See the instructions for balancing the sample tubes on the next page.

6) Add 200 µl ethanol (96-100 %) to the sample, and mix again by pulse-vortexing for 15 seconds. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.

7) Carefully apply the mixture from step 6 to the QIAamp Mini spin column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 minute. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate.*

8) Carefully open the QIAamp Mini spin column and add 500 µl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 minute.
Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the collection tube containing the filtrate.

9) Carefully open the QIAamp Mini spin column and add 500 µl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 minutes.

10) Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube (not provided), and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 200 µl Buffer AE or distilled water. Incubate at room temperature (15-25 °C) for 1 minute, and then centrifuge at 6000 x g (8000 rpm) for 1 minute.

11) Discard the spin column and seal the 1.5 ml tube containing the genomic DNA.

Taqman-based mutation/single nucleotide polymorphism detection using QuantStudio PCR Real-Time PCR system

You will amplify your extracted DNA samples, and the 4 control DNA samples using the QuantStudio Real-Time RT-PCR system. The assay used contains two probes, in addition to forward and reverse primers. One probe (VIC-labelled) detects the normal allele, and the other probe (FAM-Labelled) detects the mutant allele. If both are detected, then the sample is heterozygous.

1) Each reaction (10 µl volume) should contain the following: Water 3.5 µl
2x Taqman mastermix 5 µl
20x Primer/probe mix 0.5 µl DNA 1 µl

However, reactions are set up in duplicate. It is therefore easier to make a mastermix as follows:

For 8 samples you need to make enough mastermix for 10 samples (4 DNA extractions, 4 controls, + water blank + spare), and since we are using duplicates, you must make enough mastermix for 20 reactions:

Water 70 µl (3.5 µl x20)
2x Taqman mastermix 100 µl (5 µl x20)
Primer/probe mix 10 µl (0.5 µl x20)

3) Aliquot 18 µl of this into 9 tubes (labelled for the 4 DNA samples, 4 controls, water blank).

4) Add 2 µl of sample (DNA or water).

5) Spin the tubes briefly.

6) Load 9.5 µl into two adjacent wells of a 96-well PCR plate, making sure you mix the DNA with the mastermix as you pipette.

7) Load the plate onto the QuantStudio system using parameters for Taqman reagents and SNP detection.

Attachment:- DNA genotyping Lab.rar