Biol 3334
Analysis of Phenylthiocarbamide Taste Alleles
1
Introduction
The ability to taste phenylthiocarbamide (PTC, Figure 1) is generally considered to be a dominant trait in
the human population. In 2003, the gene TASR38 which encodes a receptor protein on chromosome 7
was identified (Kim et al., 2003). Three distinct single nucleotide polymorphisms (SNPs) were identified
that correlate with the ability or inability to taste the compound PTC. The main SNP found to correlate
strongest with the taster/non-taster phenotypes was a transversion of C229 to G which changes the 49th
codon from CCA encoding proline to GCA which encodes alanine. The P49A change corresponds to the
non-tasting allele (i.e. the tasting allele has a proline while the non-tasting allele has an alanine at position
49). Two other SNPs produce the changes A262V and V296I. Based on these amino acid changes, the
taster allele sequence is referred to as PAV for the amino acids found at positions 49,
262, and 296 respectively. Likewise the non-taster allele sequence is called AVI representing changes at all three positions. The set of SNP sequences on each chromosome are referred to as a haplotype. Individuals that are homozygous tasters would
have two copies of the haplotype PAV (i.e. their genotype would be PAV/PAV);
whereas a non-taster would most likely have the genotype AVI/AVI. The taster haplotypes is dominant, so individuals with at least one copy of this allele usually taste
Figure 1. Structure of
the PTC compound. Because each of the SNPs can be individually different, there phenylthiocarbamide
are eight different haplotypes and 36 different genotypes. Each of these different
genotypes can vary in their ability to confer tasting versus non-tasting phenotypes.
Using PCR, we can detect the base change at one of these SNP positions, position 229. By purposefully
designing the DNA sequence of the forward oligonucleotide primer (TASR38F) so that it changes one
base of the target sequence (see * figure 2 and methods) a novel restriction sight can be created in the
PCR product produced from the non-taster allele. By digesting the PCR product with the restriction enzyme HaeIII which cuts at the sequence GGCC, PCR product made from the taster allele can be distinguished from PCR product made from the non-taster allele. HaeIII digestion will cut the taster PCR
product into fragments of 44 and 177 bp, while the 221bp non-taster PCR product having the sequence
GGGC will remain uncut.
Method
PTC haplotype detection PCRs were set up using DNA previously isolated by the chelex method (BioRad, CA) per the manufactures instructions. The sequence of the forward primer (TASR38F) is
5’-CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG-3’ and the sequence of the
reverse primer (TASR38R) is 5’-AGGTTGGCTTGGTTTGCAATGATC-3’. Amplification with these
primers produces a 221bp amplicon product. The G residue at position 43 in the primer is a mismatch to
the genomic DNA and will generate the sequence GGGCA (nt 227-231, figure 2) in a DNA with the nontaster haplotype SNP at this position (P49A). In DNA with the taster haplotype at this position, the PCR
product will have the sequence GGCCA (nt 227-231). This creates a HaeIII recognition site which will
cut at position 44 of the PCR product. PCRs were assembled using 11 μl of genomic DNA, 12.5 of 2X
Brilliant II SYBR green PCR master mix (Stratagene, CA), and 10 pmole each forward and reverse primers in a 25 μl PCR. Reactions were carried out in a Stratagene Mx3005 RT-PCR machine with a reaction profile of 94C for 30sec, 55C 30sec, 68C 30sec for 40 cycles. To determine the haplotypes present
was digestion with HaeIII was performed in 10 μl reactions containing 5 μl of each PCR product and 10U
HaeIII (Carolina Biological) in 1X reaction buffer supplied by the manufacturer. DNA fragments were
separated following HaeIII digestion by electrophoresis on 8% polyacrylamide, 1X TAE (40mM Tris acetate pH 7.5, 10mM EDTA) gels and visualized by staining with 50 μg/ml ethidium bromide in water.
Biol 3334
Analysis of Phenylthiocarbamide Taste Alleles
1 CCTTTCTGCACTGGGTGGCAACCAGGTCTTTAGATTAGCCAACTAGAGAAGAGAAGTAGA
61 ATAGCCAATTAGAGAAGTGACATCATGTTGACTCTAACTCGCATCCGCACTGTGTCCTAT
M L T L T R I R T V S Y
121 GAAGTCAGGAGTACATTTCTGTTCATTTCAGTCCTGGAGTTTGCAGTGGGGTTTCTGACC
13 E V R S T F L F I S V L E F A V G F L T
* g

181 AATGCCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCAGCCACTGAGCAAC
33 N A F V F L V N F W D V V K R Q P L S N
A
241 AGTGATTGTGTGCTGCTGTGTCTCAGCATCAGCCGGCTTTTCCTGCATGGACTGCTGTTC
53 S D C V L L C L S I S R L F L H G L L F
301 CTGAGTGCTATCCAGCTTACCCACTTCCAGAAGTTGAGTGAACCACTGAACCACAGCTAC
73 L S A I Q L T H F Q K L S E P L N H S Y
361 CAAGCCATCATCATGCTATGGATGATTGCAAACCAAGCCAACCTCTGGCTTGCTGCCTGC
93 Q A I I M L W M I A N Q A N L W L A A C
421 CTCAGCCTGCTTTACTGCTCCAAGCTCATCCGTTTCTCTCACACCTTCCTGATCTGCTTG
113 L S L L Y C S K L I R F S H T F L I C L
481 GCAAGCTGGGTCTCCAGGAAGATCTCCCAGATGCTCCTGGGTATTATTCTTTGCTCCTGC
133 A S W V S R K I S Q M L L G I I L C S C
541 ATCTGCACTGTCCTCTGTGTTTGGTGCTTTTTTAGCAGACCTCACTTCACAGTCACAACT
153 I C T V L C V W C F F S R P H F T V T T
601 GTGCTATTCATGAATAACAATACAAGGCTCAACTGGCAGATTAAAGATCTCAATTTATTT
173 V L F M N N N T R L N W Q I K D L N L F
661 TATTCCTTTCTCTTCTGCTATCTGTGGTCTGTGCCTCCTTTCCTATTGTTTCTGGTTTCT
193 Y S F L F C Y L W S V P P F L L F L V S
721 TCTGGGATGCTGACTGTCTCCCTGGGAAGGCACATGAGGACAATGAAGGTCTATACCAGA
213 S G M L T V S L G R H M R T M K V Y T R
781 AACTCTCGTGACCCCAGCCTGGAGGCCCACATTAAAGCCCTCAAGTCTCTTGTCTCCTTT
233 N S R D P S L E A H I K A L K S L V S F
841 TTCTGCTTCTTTGTGATATCATCCTGTGCTGCCTTCATCTCTGTGCCCCTACTGATTCTG
253 F C F F V I S S C A A F I S V P L L I L
901 TGGCGCGACAAAATAGGGGTGATGGTTTGTGTTGGGATAATGGCAGCTTGTCCCTCTGGG
273 W R D K I G V M V C V G I M A A C P S G
961 CATGCAGCCATCCTGATCTCAGGCAATGCCAAGTTGAGGAGAGCTGTGATGACCATTCTG
293 H A A I L I S G N A K L R R A V M T I L
1021 CTCTGGGCTCAGAGCAGCCTGAAGGTAAGAGCCGACCACAAGGCAGATTCCCGGACACTG
313 L W A Q S S L K V R A D H K A D S R T L
1081 TGCTGAGAATGGACATGAAATGAGCTCTTCATTAATACGCCTGTGAGTCTTCATAAATAT
333 C *
1141 GCC
Figure 2. Nucleotide sequence of the TASR38 gene. The encoded amino acids are shown
beneath the nucleotide sequence. The non-taster allele SNP at position 229 is indicated.
This transversion generates an alanine codon at position 49. The positions of the forward
and reverse priming sites are underlined with arrows. The * above the A at position 227
indicates the position changed to a G in the PCR product.
2