APOG is a hypothetical polymorphic short tandem repeat (STR) locus in humans wit
ID: 226069 • Letter: A
Question
APOG is a hypothetical polymorphic short tandem repeat (STR) locus in humans with a repeating unit of [CAGA]. The polymorphic region is shown below as a box with 25 bp of flanking DNA sequence on each side. The n represents the number of times [CAGA] on the top strand is repeated (the polymorphic part of STR locus). (a) You plan to use PCR to genotype individuals for the APOG locus. If PCR primers must be 18 nucleotides long, design a pair of primers to amplify the APOG locus? Design the primers such that they are immediately adjacent to the [CAGA] repeat (i.e. don't worry about optimizing annealing temperature, GC content, etc. -everybody's primer sequences should be the same). (b) Consider APOG alleles with 9 and 7 copies of the repeating [CAGA] unit. Using the primers you have designed, what will be the sizes in base pairs of the PCR products for each allele? In other words, if run on an electrophoresis gel, what would be the sizes of the two bands? (c) There are five known alleles of APOG with 14, 11, 10, 9 and 7 copies of the repeating unit. How many possible human genotypes are there for these alleles, and what are they? (d) If one parent is heterozygous for the 14 and 9 alleles of the APOG locus and the other parent is heterozygous for the 9 and 7 alleles, what are the possible genotypes of their offspring for this locus, and in what proportion will they be found?Explanation / Answer
(a) You plan to use PCR to genotype individuals for the APOG locuc. If PCR primers must be 18 nucleotides long, design a pair of primers to amplify the APOG locus? Design the primers such that they are immediately adjacent to the CAGA repeat.
Answer: The primer length is 18 nucleotides and is immediately adjacent to the [CAGA] repeat. The forward primer is derived from the top sequence reading from 5’ towards 3’ direction. It will be 18 nucleotides before the repeat i.e.
Forward primer: 5’- GTGATCTCCTTTAGCTTC-3’
The reverse primer is derived from the bottom sequence (complimentary strand). The sequence immediately after the repeat will be used and since its read in the 5’ to 3’ direction the sequence will be reverse of the sequence given
Reverse primer: 5’- TCACGTAACGAATGGTAC-3’
(b) Consider APOG alleles with 9 and 7 copies of the repeating [CAGA] unit. Using the primers you have desgined, what will be the sizes in base pairs of the PCR products for each allele? In other words, if run on an electrophoresis gel, what would be the sizes of the two bands?
Answer: The size of the band will be the size of repeat plus the size of the two primers. The combined size of the two primers will be 36 (18+18), so
Size of allele with 9 copies = (9*4) +36 = 72 bp
Size of allele with 7 copies = (7*4) + 36 = 64 bp
c) There are five known alleles of the APOG with 14, 11, 10, 9 and 7 copies of the repeating unit. How many possible human genotypes are there for these alleles, and what are they?
Answer: Since each genotype will have two alleles ( humans have two sets of chromosomes, so two alleles for each gene) and there are five possible alleles we can have a total of 15 human genotypes are possible as below
14/14, 14/11, 14/10, 14/9, 14/7, 11/11, 11/10, 11/9, 11/7, 10/10, 10/9, 10/7, 9/9, 9/7, 7/7
(d) If one of the parents is heterozygous for the 14 and 9 alleles of the APOG locus and the other parent is heterozygous for the 9 and 7 alleles, what are the possible genotypes of their offspring for this locus and in what proportion will they be found
Answer: Each offspring will get one allele from each parent. Following combinations will be possible
Parent 1 alleles = 14 and 9 combines with 9 and 7 i.e parent 2 allele i.e
14 or 9 X 9 or 7, so
Possible genotypes will thus be 14/9, 14/7, 9/9 and 9/7. All the offsprings will be in equal proportions i.e there is equal chance for the offsprings to have any of the four genotypes!