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Essay On Whole Genome Sequencing

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2.0 Materials and Methods
Whole genome sequencing (WGS) is one of the current methods used to obtain the entire genetic composition of a particular patient. Once the sequence of DNA has been obtained the information within the patient’s genome is compared to a reference genome so that potentially pathogenic mutations can be identified. Regions of DNA that are of interest are validated following the procedure outlined below.

Primer design

Specific DNA loci were obtained from analysts in the form of Excel spreadsheets. These coordinates were visualized using the genome browser build 19 on the UCSC website. The DNA sequence that was taken was approximately 900 bp on either end of the region which was to be amplified. The DNA sequence which
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This extension period was set for 7 minutes. 35 cycles were run when amplifying particular regions of DNA obtained from probands and their respective families. Note primers were tested with controls using the hotstart PCR conditions before primers were run with the patient’s DNA.

Visualizing PCR product

Agarose gels were run to determine whether amplification of the targeted region of DNA was successful. 1% gels were made using 50mL of 1X TAE buffer and 0.5g of agarose. 2.5mL of Ecosafe dye was added to the gel before mixing and then cooling in a gel rig. 1.0mL of loading dye was mixed with 4.0mL of DNA from PCR tubes. DNA samples were then loaded in their respective wells along with 5mL of 1kb ladder to approximate band sizes during visualization of the gel. Gels were visualized using UV light set at a wavelength of 302nm. Each gel was exposed to UV light for 10 seconds before the image was captured with an Azure gel imaging system.

Purification

PCR tubes were ready for purification when a single DNA band was present per lane on the agarose gel. If multiple bands were obtained in a single lane the PCR process and gel visualization process was repeated however, the denaturation temperature was run at a higher temperature to prevent non-specific binding of the primers to the DNA template. The purification process used an Invitrogen PCR clean-up kit. 5mL of charge switch beads
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