in this is a lab were we amplified the Alu at a particular locus called TPA-25 i
ID: 200730 • Letter: I
Question
in this is a lab were we amplified the Alu at a particular locus called TPA-25 in human cheek epithelial cells through the use of PCR in a population of 28 students.
please help me with these questions listed below;
How successful were the designed primers at amplifying the TPA-25 locus? What could have contributed to a poor amplification?
Which primer set was better? What reasons can you think of that would have made it better?
Is the TPA-25 locus important? If the Alu gene is inserted in that locus, is it phenotypically neutral?
the three primers that were used in the experiment are;
Primer 1:
Forward: CCC AAG TTA AGG GTC CTG GC
Reverse: AGG GAG GAC ACC GAG TTC AT
Length of fragment is there is no insert: 224 bp
Primer 2:
Forward: TTC ACT TGC TAG AAT AAC AC
Reverse: CCA TGT AAG AGT AGA AGG AG
Length of fragment is there is no insert: 338 bp
Primer 3:
Forward: GTA AGA GTT CCG TAA CAG GAC AGC T
Reverse: CCC CAC CCT AGG AGA ACT TCT CTT T
Length of fragment is there is no insert: 100 bp
Legend:
1: 10l of DNA Ladder
2: PCR reaction mix with primer 1, epithelial cheek cell DNA from Michelle.
3: PCR reaction mix with primer 2, epithelial cheek cell DNA from Michelle
4: PCR reaction mix with primer 3, epithelial cheek cell DNA from Michelle
5: PCR reaction mix with primer 1, epithelial cheek cell DNA from Mowa
6: PCR reaction mix with primer 2, epithelial cheek cell DNA from Mowa
7: PCR reaction mix with primer 3, epithelial cheek cell DNA from Mowa
the gel above is the one that I and my partner ran.
Primer 1:
Forward: CCC AAG TTA AGG GTC CTG GC
Reverse: AGG GAG GAC ACC GAG TTC AT
Length of fragment is there is no insert: 224 bp
Primer 2:
Forward: TTC ACT TGC TAG AAT AAC AC
Reverse: CCA TGT AAG AGT AGA AGG AG
Length of fragment is there is no insert: 338 bp
Primer 3:
Forward: GTA AGA GTT CCG TAA CAG GAC AGC T
Reverse: CCC CAC CCT AGG AGA ACT TCT CTT T
Length of fragment is there is no insert: 100 bp
Legend:
1: 10l of DNA Ladder
2: PCR reaction mix with primer 1, epithelial cheek cell DNA from Michelle.
3: PCR reaction mix with primer 2, epithelial cheek cell DNA from Michelle
4: PCR reaction mix with primer 3, epithelial cheek cell DNA from Michelle
5: PCR reaction mix with primer 1, epithelial cheek cell DNA from Mowa
6: PCR reaction mix with primer 2, epithelial cheek cell DNA from Mowa
7: PCR reaction mix with primer 3, epithelial cheek cell DNA from Mowa
the gel above is the one that I and my partner ran.
Explanation / Answer
Alu elements are short interspersed elements (SINEs), which are 300 nucleotides in length.
All the three primer sets targeting TPA gene without insert.
In this experiment, oligonucleotide primers flanking the insertion site will be used to amplify a segment of the TPA gene, The expected product of the PCR reaction will be approximately 400- bp fragment when TPA-25 is present, and a 100-bp fragment when it is absent.
So I think primer 2 is better as the amplicon size of the product (without insert) is 338 bp, which is greater than amplicon synthesized by other 2 primer sets.
Primer 1 and 3 can also be useful and it depends on the TPA-25 locus in the genome.
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