Kuliah

Elektif Genetik 2020

Gene, PCR primers and understanding genetic variants
Galuh DN Astuti
07 September 2020

Read me first

• The goal of this practical work is to know the basic principles of gene and its structure,
designing primers for standard PCR and genetic variant nomenclature.
• Questions are in italics and designated by “Q.”
• Not all questions have definite answers but are meant to help you think about the
data/information that is presented.
• The blue boxes contain hints that are available for some questions.
• There are quick references of websites and tools used during this workshop (see list of
contents)
• Useful links are listed in the last page of this manual
• You may ask questions during the workshop.

Case
A 35-year-old woman referred to an ophthalmologist with a 6-month history of glare and
painless diminution of vision in both eyes. Her best-corrected visual acuity was 20/20 in the
right eye and 20/60 in the left eye. White "bread crumb" opacities with "moth-eaten" centers
were observed in the superficial and mid-corneal stroma (A, B). These were interspersed with
star-shaped, spiny, or icicle-shaped deposits as seen in anterior segment optical coherence
tomography (C, D). The patient stated that there are more affected individuals in her family.

Based on the ophthalmologic assessment, the ophthalmologist diagnosed this patient with
Avellino Corneal Dystrophy also known as combined granular-lattice dystrophy, or granular
corneal dystrophy (GCD) type 2.

Part1 : Gene hunting (15 minutes)

Family pedigree

In the pedigrees, squares represent male individuals and circles represent females individuals.
An arrow indicates the proband. Affected individuals are represented by the filled symbols.

Online Mendelian Inheritance in Man (OMIM) is a freely available, continuously updated catalog of
human genes and genetic disorders and traits, with a particular focus on the gene-phenotype
relationship. As of 28 June 2019, approximately 9,000 of the over 25,000 entries in OMIM represented

phenotypes; the rest represented genes, many of which were related to known phenotypes.

URL: https://www.omim.org/

Open a new window on your internet browser and go to OMIM and read the description about
the disorder.

Q1. Which gene is relevant for this disease?

Q2. Based on the pedigree, what is the possible mode of inheritance of the disease? Explain your
reason(s).

Q3. Based on the information in OMIM, is there any common mutations or hotspot mutations in
this gene?

Part2: Reading the sequencing result (45 minutes)

You have performed PCR using your primers and thereafter Sanger sequence them. You get your
results back in .ab1 file format. Use FinchTV to visualize your data

Step 1 Download the .ab1 chromatogram data
(file amplicon1_F.ab1 and amplicon1_R.ab1)

Step 2 Open FinchTV

Step 3 Load the file amplicon1_F.ab1 by clicking “File”and then click “Open”

And then find the location where your file is located

Now you will see the chromatogram image of file amplicon1_F.ab1

Step 4 You can adjust the height and width of the peaks using vertical and horizontal Scale, so
that it is easier to read

Hint: In a DNA chromatogram, each DNA base is represented as a peak of a different color
i.e. A: green peaks, G: black peaks, T: red peaks, C: blue peaks. You can see single nucleotide
variant or small insertion/ deletion from this chromatogram.

Step 5 Click on position 10 and look at the information on the left lower pane (Selected base
A:10 (Q6))
Hint: The position information is on the top part below the nucleotide sequence

Step 6 Click on position 20 and look at the information on the left lower pane

Q4. Compare the information from step 5 and 6 and what do you think “Q” means?

Step 7 Click on “Wrapped view” option to see the entire sequence

Step 8 Click on “Chromatogram info” to see the entire nucleotide sequence. Select the entire
sequence and click “

Now we know and able to read the entire sequence, but how to detect whether there is any
variants/mutations?

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Step 9 Block from position 160 to 180, copy then paste to a separate file

Step 10 Block from position 160 to 180, and then click at “Reverse Complement” (look at the
arrow). Copy and then paste to a separate file

Step 11-13 is the same as step 3, 9 and 10 but using different file (amplicon1_R.ab1, instead of
amplicon1_F.ab1)

Step 11 Open the file amplicon1_R.ab1 by clicking “File”and then click “Open”
Step 12 Block from position 341 to 361, copy then paste to a separate file
Step 13 Block from position 190 to 210, and then click at “Reverse Complement” (look at the
arrow). Copy and then paste to a separate file

Q5. Compare the DNA sequence from step 9, 10, 12 and 13 and highlight part that are similar
and part that are different.

Q6. As you know from previous lectures, that there are four different nucleotides which compose
DNA, i.e., A,T,C and G. So what does “R” and “Y” mean in those sequences?

Hint: Look at the number of peak and its colors

Part3: Locating the mutation in the human genome reference (30 minutes)
From part 2 of this workshop, we are able to read the DNA sequence. However, do you know in
which part of the human genome this sequence is located?
This part of workshop will help you to answer this question by using some publicly available
website
Step 14 Open UCSC genome browser website, click “Tools” and then “ Blat”
BLAT on DNA is designed to quickly find sequences of 95% and greater similarity of length 25
bases or more

Step 15 Copy and paste your sequence into the empty box, select assembly GRCh37/hg19 and
then click submit

Step 16 You will see some matches, click on “details” of the highest matching percentage

Step 17 Check the side by side alignment. The reference (normal human genome) is underlined
by blue lines and your sequences is underlined by red lines.

Q7. Compare the two variants and circle where the variant is located.

Q8. Look carefully the page after the last step. Can you find in which chromosome the variants is
located? And which genomic position exactly of that chromosome?

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