Using the in-Silico PCR tool in the UCSC Genome Browser
In this tutorial, we will explore the UCSC Genome Browser’s In-Silico
PCR tool. This tool allows you to predict the sequence and size ofproducts that would result from a polymerase chain reaction
experiment using primers you may wish to use. You can see that there’sa link to the tool directly on the main page, at genome.ucsc.edu.

Let’s go to the Genome Browser and “Reset all user settings” because touse the in-silico PCR tool we must first have some primers to input intothe tool, and we will navigate to the Genome Browser to find them.

[0:40 Set the Genome Browser to hg19 defaults]

Let’s go to the hg19 assembly, as that’s still the most commonly usedhuman assembly, and use the “go” button to navigate directly to the
Genome Browser graphic. Below the graphic, find the “hide all”
button to turn off the numerous data tracks visible and set the “UCSCGenes” track to “pack.”[1:08 Navigate to a gene (CYP2D6) to obtain DNA for primers]

Let’s navigate to the CYP2D6 gene, choose it from the drop downsuggestion menu, and hit the “go” button to navigate there. You see weare on chromosome 22. The reason for choosing this gene is that there
is a very obvious differentially spliced exon in the middle of the gene,which allows us to use the In-Silico PCR tool in both of its modes, which
are essentially using either DNA or spliced RNA as template so that you
can see the results of the experiment both ways.
[1:46 Get DNA with exons annotated]

So, let’s zoom into the three exons with the differentially expressed exon
in the middle of the screen, and go to the “View... DNA” pulldown in thebluebar menu to extract some DNA to use as primers.

“Extended case/color options” at the bottom of the screen
allow us to use the UCSC Genes track to decorate the DNA output andmake it more obvious where the exons are. Let’s select “lowercase”

for the default so that we can toggle the case to capitals where the UCSC
Genes track indicates the presence of the exons. We’ll use 255 in theBlue box in the RGB color selector so that the capital letters are blue andmuch more obvious. Then let’s “submit”.
[2:40 Obtain primers for PCR from exons]

You can see here that we have three blue exons in capital letters and weobtain our first primer by copying from the top, the 5’ end of thesequence, of the upper strand of this exon. We will get a chunk of DNA
large enough to serve as a primer and paste it into a text file so that wecan use it twice in our demonstration. Now we’ll grab a chunk of DNA
from the other end of this region, from the third exon, and save it also toa text file. Then let’s return to the Genome Browser, and select “In-SilicoPCR” from the “Tools” drop down menu in the bluebar at the top.

[3:25 Use primers to predict amplicon size from genome assembly]

We will paste the reverse primer that’s still in our copy buffer into the“reverse primer” box, and we will flip the orientation of the reverse
primer so that the 3’ ends of our two primers are facing each other.
We’re essentially taking the second primer and using it on the lower
strand, or reverse strand, of the DNA template.

Going now to the text file, we will grab our forward primer, and paste it
into the “forward primer” box in the In-Silico PCR tool. Notice that the“target” is set to the default, “genome assembly,” which is essentiallyusing DNA as a template, with any introns included. Now hit “Submit”.
[4:12 Observe amplified DNA in Genome Browser]

You’ll notice in the results that we actually have two amplicons
predicted by the tool. One of them is 1085 and the other is 1108 basepairs. Let’s click into the top one, the 1085 bp amplicon, and we’ll noticethat we are on the genome on chromosome 22 but we are at CYP2D7P1,a pseudo gene that apparently closely resembles the 2D6 gene where
we got our primers. In fact, you’ll notice that there’s a 1 base pairmismatch annotated here by a red tick mark, near the left end of thisannotation.

Let’s zoom out by a factor of 10X, and then another factor of 10X andwe’ll observe that there’s a second amplicon to the left of this one that
represents the CYP2D6 gene that we began with. In fact, the gene nameis still highlighted from our having typed it into the position box when
we navigated to this region of the genome. You’ll see that this illustrates
one of the primary purposes of having the isPCR tool and one of thefeatures of the tool: It represents an opportunity to confirm that yourprimers amplify uniquely in the genome and will not amplify at alocation elsewhere in the genome that might not have been known ormight not have been expected when the primers were originally chosen.
[5:32 Use primers to predict amplicon size from RNA template]

So let’s go back to the isPCR tool, and reset the target to “UCSC Genes”,
which is the In-Silico equivalent of having spliced mRNA as a template,
and we will paste the forward primer, still in our copy buffer, into theforward primer box. Then we’ll go back to our text page, grab thereverse primer, paste the reverse primer into its box, and notice that theflip-reverse-primer checkbox is still selected. Then hit “submit.”

Now on this results page you’ll notice that we have multiple results. We
did expect to find at least the CYP2D6 and the CYP2D7 pseudogene
because of our earlier result with these primers, and we’ll also noticethat we have multiple hits for each of these. Notice also that theCYP2D6 amplicons are 315 bp and 468 bp in length.
[6:36 Observe RNA results in Genome Browser]

Let’s click into the upper CYP2D6 amplicon, navigate to the GenomeBrowser, and see that we actually have three annotations of two
different types, representing the three isoforms of the gene, each as aseparate amplification product. One includes the intervening exon andthe other two do not. By zooming out by a factor of 10X, we see that theCYP2D7 pseudo gene actually has four predicted amplicons using theprimers that we selected, corresponding to the four isoforms of thisgene.

So that takes you through our tour of the isPCR tool in the UCSC Genome
Browser, showing you the two different modes of operation, one using
DNA and one using spliced RNA as the target. Note that the UCSC genes
option, which is essentially the RNA amplification mode, is only
available for human and mouse assemblies.
Thanks for watching and thanks for using the UCSC Genome Browser.