Module 2, Lab 05 - GAPDH Nested PCR

Thale cress, Arabidopsis thaliana

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Thursday (Round 1)

Today we started the exercise in which students will learn the basics of nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in the thale cress (Arabidopsis thaliana), the model organism of plants. Some people call it "the fruit-fly of plants".

GAPDH is an enzyme in charge of catalyzing one of the reactions in glycolysis. There are several nuclear genes that encode GAPDH isomers (proteins with different amino acid sequences but with the same function), and we are targeting the gene GAPC in the A. thaliana genome. We ran a first round of PCR, with our initial primers, and tomorrow we will run the second run, with the nested primers.

Friday (Round 2)

Today we ran the second round of PCR, using the nested primers.

In order to do so the first round primers were degraded adding an exonuclease to the amplified samples and incubating for 15 minutes at 37ºC. Then the exonuclease was denatured by incubating the samples at 80ºC for 15 minutes.

Finally, the Arabidopsis genomic DNA was diluted (1:50) and used to set up the second run of PCRs.

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Lecture, chapter 6 - Transcription

We started chapter 6 on Transcription.

We discussed general introductory issues on the process of transcription, including terminology of components and processes (e.g. template or sense strands vs. coding or anti-sense strands, hosekeeping genes, cistrons, open reading frames [ORFs], operons, and monocistronic and polycistronic DNA).

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Lecture, Chapter 5 - DNA replication

Today we finished the DNA replication chapter.

We discussed the main differences between prokaryotic and eukaryotic chromosomes, and how the ends of eukaryotic chromosomes (telomeres) are repaired by the action of telomerases (three scientists were awarded the Nobel Prize in physiology or medicine, in 2009, because of their research on telomerases).

Among the differences between prokaryotic and eukaryotic DNA replication we mentioned how in eukaryotic chromosomes have multiple replication origins, and in their DNA replication process there are two DNA polymerases and a few proteins that are not found in the prokaryotic DNA replication.

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Lecture, chapter 5 - DNA replication

Today we covered most of chapter 5, on DNA replication. We talked about the replication fork and the replisome (all the enzymes that are part of the replication fork), including the difference in between how the leading and lagging strands are synthesized.

We also mentioned how important DNA looping is for the lagging strand to be synthesized. This video illustrates such phenomenon beautifully:

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