PRACTICAL 5

Overview
 Restriction Digest
 SNPs and Synonymous vs non-synonymous mutations
 Download a free program called MEGA or use it from the Virtual Desktop (see
instructions below)

AIM

This week you will genotype your worms. Your PCR from last week has been run, so we
now have a sample of tubes for each class that should now contain a PCR-product that spans
the strain specific SNP that differentiates between the two different strains used to set up the
experiment. We detect which allele is present by restriction digestion. Restriction
endonucleases are enzymes that recognise a specific DNA sequence (usually either 4 or 6
base pairs in length) and cleave (cut) the DNA at that point. Consequently, a PCR product
that contains the recognition site will be digested whereas a PCR product that does not
include the recognition site (because of a polymorphism in the DNA sequence) will not
cleave. Thus, it is possible to determine which allele is present by testing the PCR product for
cleavage and hence the stain of origin of the worm you picked to that tube.

Materials and Methods: restriction digest and gel electrophoresis.

1. Your worms have been PCRed.

2. Find 2 tubes, it doesn’t matter if they are yours or not – the tubes are separated by
“Population 1” or “Population 2 – it is VERY important you take note of which
population tubes you pick up.

3. Your demonstrator will have a tube marked with “Restriction enzyme mix”

4. Add 5l of the restriction enzyme mix to your tubes

5. Put masking tape on top of your tubes with your name and the population number

6. Place your tubes in the 37oC water bath for 60 minutes for the restriction enzyme
digestion to take place.

7. When your two tubes come out of the water bath, go to one of the electrophoresis
work stations set up around the room – make sure Zoia or a demonstrator are standing
next to it.

8. Tell us whether you have population 1 or population 2 tubes

 9. Load 10l of the mix in your tubes, and give your tubes to the demonstrator.

Gel Electrophoresis

DNA naturally contains a net negative charge due its sugar-phosphate backbone, and hence
will migrate towards the positive terminal. The gel itself is a matrix of pores, size -
dependent on the concentration of agarose used to make the gel. DNA fragments move
through the gel relative to their size, with smaller fragments moving through the pores more
easily than larger fragments, which get impeded by the gel, and hence move more slowly.

Figure 5.1. Agarose gel electrophoresis method. Image taken from

https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-
science/agarose-gel-electrophoresis (accessed 29/3/2023)

1. DNA to be analysed is loaded into a well at the top of the gel.

2. A DNA ladder of known molecular weight size is also added to an additional well, which
allows estimation of your DNA sample size.

3. An electric current is passed through the gel and the surrounding buffer, which flows from
the top negative anode, towards the bottom positive cathode. As DNA is negatively
charged, both the sample and the ladder will begin to move towards the positive cathode.
The agarose acts as a sieve, preventing larger DNA molecules from passing through while
allowing smaller DNA fragments easier passage. In this way, DNA fragments of different
sizes move relative to their size: small DNA = faster; larger DNA = slower.

4. Once the gel has finished running, it is stained with a DNA binding dye, such as Gel Red.

5. The DNA bands from the sample and the ladder can now be visualized under UV light,
which can then be photographed using a special camera.
Visualising DNA as a sequence and on a Gel
We know that on chromosome V there is a region of the DNA of the worms where the strain
CB4856 has a SNP (change from A to T) which is absent from other strains.

The SNP allows the restriction enzyme DraI, which recognises the sequence TTTAAA, to
digest the DNA of any CB4857 worm but not a CX11314 worm.

The entire DNA fragment is 443bp in size and since the restriction enzyme does not cut the
DNA of CX11314, the size of the fragment for CX11314 = 443bp

Because CB4856 has the cut site TTTAAA, the enzyme does cut the DNA of CB4856 at
at the last T of TTTAAA, producing two DNA fragments, one 162bp in size, the other 281bp
in size (162+281 = 443).

When run on a gel, the results look as follows:

Today we will look at a few SNPs which occur on Chromosome I of C. elegans

You need access to a program called MEGA XI. Mega is free to download or you can access
it from the Virtual desktop.

If you have a Mac you are better off using the Virtual Desktop

Downloading Mega:

Mega is free to download and works on both PCs and Macs.

Go to: www.megasoftware.net

Select the appropriate operating system from the drop down menu then select MEGA 11 and
press “Download”.

Using the virtual Desktop:

Go to: https://www.latrobe.edu.au/students/support/it/teaching/myapps

From the start menu, navigate to a folder called “Mega X”

Click on it and select MEGA X

 Open Mega.
  Click on Align
 Edit/Build Alignment
 From the pop up window select: Retrieve sequence from file (you might need to drag
the pop-up window to show all 3 options)

 Click OK
 A search window will open – go to your practical 5 folder, and find a document called
“Chromosome 1 Alignment”
 Click on it
 You should have a screen like this:

We will look at 4 worm strains:

N2 – isolated from mushroom compost in an urban garden in Bristol in 1951
CB4856 – isolated from a pineapple plantation in Honolulu, Oahu, Hawaii in 1972
JT11398 – isolated from a worm bin used for compost in Lake Forest Park, Seattle 2003
JU258 – isolated from soil from an orchard in Madeira Portugal in 2001

Click on

In the pop up window type TTTAAA: (not case sensitive)

Press ok

You should see a region highlighted in yellow:

Scroll along the length of the sequences – how many times does the TTTAAA cut site appear
in the different strains?

Worm strain # TTTAAA cut sites

N2
JU258
JT11398
CB4856

Using the site # count at the bottom:

Determine the position along the DNA where the enzyme cuts (count at the last T of
TTTAAA).

How many and what size DNA fragments would result? Remember, the entire uncut DNA
fragment is 357 bp long
strain DraI cut site Fragments
CB4856
JU258
JT11398
N2

 Click on Data
 From the drop down menu select “Phylogenetic analysis”
 A window will pop up asking:

 Click “YES”
 Another pop up window will appear very quickly and then it will close.
 Minimise your Alignment Explorer window, and return to the MEGA opening page.
 You should ow have 3 little icons visible:

Click on the
A new window will open:
The data are now presented in a slightly different way

Along the top, the bases are organized in triplets (codons). Underneath each base in the
codons is either a dot or a base. A dot means the base is the same as in the codon, a base
means it’s different

For example:

Look at the first codon shown on picture: TAA

N2 and JT11398 both have a dot under the first A

JU256 and CB4856 have a T, so the codon becomes TTA rather than TAA.

Because the SNP is in the 2nd codon position, this is a non-synonymous change which will
result in a different amino acid being made.

N2 and JT11398 make TAA which is actually a Stop codon and is designated *

JU256 and CB4856 make TTA which codes for Leucine (L)

Synonymous and non-synonymous mutations

Figure 7-24b Herron and Freeman 2014

Remember: vast majority of 3rd position mutations are synonymous (do not
change the amino acid)

A few first position mutations are synonymous, but ALL 2 nd position mutations
are non-synonymous and result in a different amino acid being made.

Amino acid changes (see next page for Amino acid table)

1. The base change at position 217 falls at the first base on a codon coding for an amino
acid.
1.1 Does the SNP cause a change in the amino acid?
1.2 What is the amino acid in CB4856and JU258?
1.3 What i is the amino acid in JT11398 and N2?

2. The base change at position 240 falls at the third base on a codon coding for an amino
acid.
2.1 Does the SNP cause a change in the amino acid?
2.2 What is the amino acid in JU258?
2.3 What i is the amino acid in CB4856, JT11398 and N2?

3. The base change at position 249falls at the third base on a codon coding for an amino
acid.
3.1 Does the SNP cause a change in the amino acid?
3.2 What is the amino acid in CB4856 and JU258?
3.3 What is the amino acid in JT11398 and N2?
Summary table
strain # synonymous changes # non-synonymous changes
N2
JU258
JT11398
CB4856
 https://www.researchgate.net/figure/Codon-tables-with-the-amino-acids-encoded-
according-to-different-properties-a-The_fig1_337736408