Question

Fred had conducted a PCR experiment for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. This is a house-keeping gene. He was searching the following primers from the following sequence:

1 ccgtattcag cattctatgc tctcaagtta tgaaacagga aatgatgacc tcctgaactt

61 gaggcagttt aactactact ttttttaaaa aggcaccaag atacttacaa aaacattttt

121 cttgttttgt ttctccatgg tttgagttta cttttaaaac tttcttttca ccagctattt

181 tggagattaa tctaacaaaa aacatgaaac ttaaatatat tttggaaatc taaattatac

241 ttagagactt aaatacattt tgctgatgac tggttacaat acagttacag actaggtata

301 tgttaaattt gaataaaaag ttattaaagc attaatcttt ttcctttcgc aaaacaagtt

361 caccaccatg tgaaataatt tcaaattaat gcataagatg tttcttccat ttacaaccac

421 aacgattctt ctgtaagtca agctcctacc attcatgctg acatttaggt agaaatttga

481 ctgttaaaaa atatgagctt catttaaact cacctttggt caatccctgg gatttgcttt

541 caaacataaa gatcaccaca aagtattaaa gaacaggctc ttagcacagc aaaacttgta

601 aaggataaaa tcattcatcc ttgcctctca gacaatgcct ggatccctaa agagacaatc

661 catttccaag actgacagcc ccagagtgtg tatccaattg aatatcgcga tgagtttatt

721 cgtcttgact ggaatttggt agtaagagaa ggaacatcca agtataagta agggctggcc

781 taaatgatac cccaccgtgt gaggtgaccg catcttcttg tgcagtgcca gcctcgtctc

841 atagacaaga tggtgaaggt cggtgtgaac ggatttggcc gtatcggacg cctggttacc

901 agggctgcct tctcttgtga caaagtggac attgttgcca tcaacgaccc cttcattgac

961 ctcaactaca tggtctacat gttccagtat gactctaccc acggcaagtt caacggcaca

1021 gtcaaggctg agaatgggaa gctggtcatc aacgggaaac ccatcaccat cttccaggag

1081 cgagatcccg ctaacatcaa atggggtgat gctggtgctg agtatgtcgt ggagtctact

1141 ggcgtcttca ccaccatgga gaaggctggg gctcacctga agggtggggc caaaagggtc

1201 atcatctccg ccccttccgc tgatgccccc atgtttgtga tgggtgtgaa ccacgagaaa

1261 tatgacaact ccctcaagat tgtcagcaat gcatcctgca ccaccaactg cttagccccc

1321 ctggccaagg tcatccatga caactttggc atcgtggaag ggctcatgac cacagtccat

1381 gccatcactg ccactcagaa gactgtggat ggcccctctg gaaagctgtg gcgtgatggc

1441 cgtggggcag cccagaacat catccctgca tccactggtg ctgccaaggc tgtgggcaag

1501 gtcatcccag agctgaacgg gaagctcact ggcatggcct tccgtgttcc tacccccaat

1561 gtatccgttg tggatctgac atgccgcctg gagaaacctg ccaagtatga tgacatcaag

1621 aaggtggtga agcaggcggc cgagggccca ctaaagggca tcctgggcta cactgaggac

1681 caggttgtct cctgtgactt caacagcaac tcccattctt ccacctttga tgctggggct

1741 ggcattgctc tcaatgacaa ctttgtgaag ctcatttcct ggtatgacaa tgaatatggc

1801 tacagcaaca gggtggtgga cctcatggcc tacatggcct ccaaggagta agaaaccctg

1861 gaccacccag cccagcaagg atactgagag caagagagag gccctcagtt gctgaggagt

1921 ccccatccca actcagcccc caacactgag catctccctc acaattccat cccagacccc

1981 ataacaacag gaggggcctg gggagccctc ccttctctcg aataccatca ataaagttc

The primers for GAPDH: 5’- aagatggtgaaggtcggtgt-3’(forward)

5’- tgactgtgccgttgaacttg-3’ (backward)

(a) In which position on the cDNA did the primer (forward) start to amplify? (1 mark)

(I think I am suppose to answer like "position x"? )

(b) In which position on the cDNA did the primer (backward) start to amplify? [Hint: It is amplifying in backward direction]

(c) What is the expected product size? Show your step.

(d) Calculate the CG content for each of the primers.

(e) Fred followed the following protocol: Samples were preheated to 94 C for 4 min and underwent 15 cycles of 94 C for 1 min, 45 C for 1 min, and 72 C for 1 min. At the end of amplification, the samples were incubated at 72 C for 10 min and stored at –20 C. However, after gel electrophoresis for 1.5 hours, he cannot find any bands on the gel and he was sure that he had load samples with dye and connected the power supply incorrect order. Describe THREE possible conditions, with explanation, that lead to the current finding.

(f) Fred’s labmate, Carlos said the failure of the experiment was due to the use of the house-keeping gene and there may be no expression in cells. Explain why Carlos was not correct.

Answer 1

a) Let's locate the primer sequence, it is here:

That is position 847

b) To locate this one we have to transform this primer into its reverse complement:

- Original sequence:   5’- tgactgtgccgttgaacttg-3’

- Reverse complement: 3'- caagttcaacggcacagtca-5'

It is located here:

That is position 1024

c) The product will be the region from the first nucleotide included in the forward primer (847) to the last nucleotide included by the reverse primer (1024). Let's do the subtraction:

1024 - 847 = 177

d) CG content is expressed as a percent, so let's count theGC occurrance:

- GC in forward: 10

- Total nucleotides in forward: 20

GC content in forward: 10/20 = 0.5 = 50%

- GC in reverse: 10

- Total nucleotides in reverse: 20

GC content in reverse: 10/20 = 0.5 = 50%

e)

1.- The annealing temperature is not the correct one. He used 45°C, but when calculating the correct annealing temperature I found 57°C, this can affect the final results

2.- Maybe he forgot to add the required reactants into the samples for the amplification to occur, maybe he forgot the dNTPs, the polymerase, or any other reactant.

3.- The primer design is problematic, sometimes we can get hairpins or primer dimer if our primer design leads to the possibility of complementing each other or folding into themselves. If that occurs, the primer are inhabilitated to go and start annealing during amplification.

f) PCR does not depend on cell expression, and even when not being expressed the gene sequence is present in 2 copies per cell, each one of this copies is going to be amplified in the PCR, completely independently from if it is being exressed or not in native conditions.