Ptc Genetics Lab Student Worksheet (Page 2 of 10) in pdf

PTC

Restriction Enzyme Digestion

What is a restriction enzyme? A restriction enzyme (an endonuclease) is an enzyme that can scan double-stranded

DNA (dsDNA) molecule for a specific sequence of base pairs known as a restriction site and then make a cut at or

near the restriction site of the dsDNA.

We used the restriction enzyme HaeIII to cut the DNA samples (the short 221 bp region of the TAS2R38 gene). HaeIII

scans for the 4 base-long restriction site “GGCC” which is only present in the TAS2R38 functional allele. In this

case, HaeIII cuts the dsDNA of the functional allele into two fragments (176 bp and 45 bp) but cannot cut the dsDNA

of the mutant allele. After incubating the DNA samples with HaeIII, the cut and the uncut DNA fragments can be

separated by size using the gel electrophoresis technique.

Based on the function of the restriction enzyme HaeIII, you know why one sample has two bands and the other has

only one, however you may be wondering why the middle one has three bands. This is because heterozygotes have

one copy of each allele: one that can be cut and one that cannot. The mutated allele cannot be cut, so the 221 bp

fragment remains intact, but the normal allele can be cut, resulting in a 176 bp fragment and a 45 bp fragment. Since

they are all present in the same sample, the final result is three fragments of distinct sizes.

The comparisons between the different types of PTC tasters and the corresponding DNA patterns on the gel are

shown in the table below:

Taster Type

TAS2R38 Genotype

HaeIII Restriction Site

DNA Pattern on Gel

Homozygous Taster

Yes, present on both

2 bands

(2 functional alleles)

functional alleles

176 bp- fragment of functional

45 bp - fragment of functional

Note: Due to the very small size of the 45 bp fragment,

this band may be faint, fuzzy, or not visible in the gel

Heterozygous Taster

Yes, present on the one

3 bands

(1 functional allele, 1

functional allele

221 bp - uncut mutant

mutant allele)

176 bp - fragment of functional

45 bp - fragment of functional

Homozygous Non-Taster

Not present on either

1 band

(2 mutant alleles)

mutant allele

221 bp - uncut mutant

Homozygous

Heterozygous

Homozygous

Taster

Non-taster

(TT)

(Tt)

(tt)

This diagram illustrates the different patterns possible

after electrophoretic separation on a 2% agarose gel. As

221 bp

you can see, a homozygous taster will have two bands,

176 bp

a heterozygous taster will have three bands, and a

homozygous non-taster will only have one band.

45 bp -

Very small band, may

be faint, fuzzy, or not visible

in the gel

Ptc Genetics Lab Student Worksheet Page 2

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