Reverse Transcriptase PCR products run on gel to be visualized. Gel image of RT-

Question

Reverse Transcriptase PCR products run on gel to be visualized.

Gel image of RT-PCR products can be seen from doing reverse transcriptase to visualze the RT-PCR products on the gel. Lane 1(100bp ladder) Lane 2 (negative control without RN)Lane 3 (negative control without reverse transcriptase), Lane 4 (contaminated DNA) thin 1300bp and thicker band 200bp Lane 5 (positive control-plasmid with a cDNA clone) 230bp.. Lane 7-RT-PCR products 210bp.

To analyze RNA gene expression. Foward 1 primer and Reverse primer that were PCR amplified a 1096 produce from genomic DNA. These primers were used to reverse trancribe and amplifiy a 150 bp. Anaylze the RT-PCR product by comparing the ladder. We should expect to see the gene transcript to be 1000 nucleotides.

Please be specific in stating the significance of the RNA products and not just only state which band is prominant. Please explain in detail. If not then do not answer question and let someone else answer. Reverse transcripase (RT-PCR) found on Lane 7 and be detailed on the significance.

Explanation / Answer

The primers were supposed to amplify a 150 bp product but the final product was 210. This is due to the random added hexamers that were also amplified increasing the final PCR product.

Random hexamer used during cDNA synthesis can attach to everywhere (so the concentration of it is quite important). Many of them can bind to a single RNA strand.

Another reason is after 1st-strand synthesis, several 1st-strand cDNAs might be attached to a single RNA strand.

So the product may be heavier than the template RNA since anyway some random hexamers are attached and some portion of RNA would have been amplified. Also, this is the reason why product size increases.

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