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Question
Preetha was practising PCR hands-on in her laboratory. She successfully isolated the gene of interest (GOI), however the quantity was too low. She desired to amplify the sequence. She conducted a PCR after which she tried to check for amplification through gel electrophoresis. She observed that all the lanes of the gel contained two bands, A and B.
Given that B was comparatively closer to the positive terminal, predict the correct alternative.
Preetha was practising PCR hands-on in her laboratory. She successfully isolated the gene of interest (GOI), however the quantity was too low. She desired to amplify the sequence. She conducted a PCR after which she tried to check for amplification through gel electrophoresis. She observed that all the lanes of the gel contained two bands, A and B.
Given that B was comparatively closer to the positive terminal, predict the correct alternative.
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Solution
The correct option is A A band is of GOI, B band is of primer-dimer
PCR, also known as polymerase chain reaction, is a technique used to amplify specific DNA sequences. This technique has three basic steps: denaturation of the double stranded DNA sequence, annealing of designed primers and extension of the newly synthesized chains.
The end product obtained after PCR is run on an agarose gel. The DNA molecules get separated based on their size in the presence of an electric field. In a gel with a specific concentration of agarose and particular pore size, a linear DNA molecule with lesser number of base pairs moves swiftly through the pores and migrates faster as compared to the one with greater number of base pairs. In other words, the rate of migration is inversely proportional to the size of a DNA molecule. Hence, the smaller DNA molecules are located close to anode (positive terminal) than the larger ones.
The primers used in PCR consist of fewer nucleotide sequences. If applied in excess in the PCR mix, the primers have a tendency to form dimers (due to complementary pairing of forward and reverse primers). These are smaller in size compared to GOI. Hence, the primer dimers are located far away from the well as compared to the GOI. GOI can be seen as bright band in the gel while primer dimer can be seen as faint band. So, B represents primer dimer while A represents the GOI.
PCR, also known as polymerase chain reaction, is a technique used to amplify specific DNA sequences. This technique has three basic steps: denaturation of the double stranded DNA sequence, annealing of designed primers and extension of the newly synthesized chains.
The end product obtained after PCR is run on an agarose gel. The DNA molecules get separated based on their size in the presence of an electric field. In a gel with a specific concentration of agarose and particular pore size, a linear DNA molecule with lesser number of base pairs moves swiftly through the pores and migrates faster as compared to the one with greater number of base pairs. In other words, the rate of migration is inversely proportional to the size of a DNA molecule. Hence, the smaller DNA molecules are located close to anode (positive terminal) than the larger ones.
The primers used in PCR consist of fewer nucleotide sequences. If applied in excess in the PCR mix, the primers have a tendency to form dimers (due to complementary pairing of forward and reverse primers). These are smaller in size compared to GOI. Hence, the primer dimers are located far away from the well as compared to the GOI. GOI can be seen as bright band in the gel while primer dimer can be seen as faint band. So, B represents primer dimer while A represents the GOI.
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