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Student 1: Emil
For the first half of this discussion board I want to focus on Northern blotting. I don’t
completely know if I understand any of the blotting techniques fully, however the best way to
know if you understand it is to try and teach it. Northern blotting and Southern blotting use
similar techniques with the main difference being the transfer of RNA opposed to DNA.
Northern blotting is a technique involving RNA that starts with gel electrophoresis that are
used to separate the molecules in the mixture by their properties like size and charge
(Griffiths, 2020 p. 334). It is important to note that the same size molecules will all end up
going the same distance on the gel (Griffiths, 2020 p. 334). This will be helpful later down the
line when reading the gel comes into play. After the molecules separate, they are transferred
to a blotting membrane where a DNA or RNA probe is incorporated into the sample. This
probe is the complementary sequence to the sample. This probe has either a radioactive atom
label or fluorescent dye. The probe is important to detect the molecule of interest in the
sample. Northern blotting techniques use RNA molecules within a specific tissue or cell type
(like blood). The main goal of northern blotting is to measure the amount of RNA expression
of specific genes. I believe that northern blotting is at the heart of transcription as it literally
runs a check to see how much RNA expression was transcribed into the original sample.
The main thing I’m struggling with in this chapter is how to read the gels to find the sequence.
This is one of the things where practice makes perfect, however I am far from that. I guess the
main thing that is tripping me up is how to identify where the bases even are. The gel I’m
going to be referring to has ddATP, ddCTP, ddGTP and ddTTP. I think it will be easier to
paint a picture of the gel before I go down the rabbit hole of trying to explain what is tripping
me up exactly. Anyways, on this gel there are markers that indicate the sequence that we have
to determine. I guess my confusion gravitates toward what base each of those represents.
What I think is; ddATP codes for A, ddCTP codes for C, ddGTP codes for G, and ddTTP for
T. While typing this I figured out where my flaw lies. I did not look at the orientation of the
gel and thought these all had to be read from bottom to top. I’m not too sure on why I was
adamant on it being this way, but I just looked back over the example and realized that the
sequence was being read from top to bottom. However, this poses a new question, when
figuring out if you are looking at the template strand or complementary, do you read one top
to bottom and the other bottom to top? I feel like this was covered, but every time I look at
these problems I find myself very confused.
Student 2 cole
In chapter 10 there were a lot of different topics from gel electrophoresis to genetic
engineering. For this chapter I am going to discuss how DNA sequencing relates to the
processes of transcription. To start off with, we first have to understand what DNA
sequencing is. DNA sequencing is best understood through dideoxy sequencing (Griffiths et
al., 2020). All of the processes that occur happen on a single DNA segment. DNA
sequencing involves a DNA primer mixture that is separated into 4 separate tubes that can be
used for gel electrophoresis (Griffiths et al., 2020). In every tube there are also
deoxynucleoside triphosphates also known as dNTPs. And there is a specific ddnTP or
dideoxynucleoside triphosphates in each tube; these include, ddATP; ddCTP; ddGTP, and
ddTTP (Griffiths et al., 2020). It is important to understand the difference between
deoxynucleoside triphosphates and dideoxynucleoside triphosphates. The ddNTPs are
modified nucleotides that add to the chain but block continued DNA synthesis (Griffiths et
al., 2020). This happens because the ddNTP does not have hydroxyl group at the 3’ location
on the sugar ribose instead they have a hydrogen atom. The hydroxyl group at the 3’ location
is used to create a phosphodiester bond between the two nucleotides and since the hydroxyl
group is not there no bond is formed (Griffiths et al., 2020). The ddNTP in the tubes creates
DNA fragments. Then gel electrophoresis is used for all of the fragments to separate them by
size from the 4 different tubes (Griffiths et al., 2020). Then the Gel Electrophoresis is read
from the bottom to top and determines the complementary sequence of the original DNA
template in the 5’ to 3’ direction. (Griffiths et al., 2020). As you can see there are a lot of
different steps that occur during DNA sequencing but these steps at heart deal with
transcription. It might not be the normal way we would think transcription occurs but DNA
synthesis is used to find the complementary strand of a DNA strand which is something that
we can relate to transcription. Because for transcription there has to be a specific DNA
sequence with respective nucleotides which can draw similarities to the process that occurs
with DNA sequencing.
Something I struggled with from this chapter was remembering which methods are tested and
are used for detecting and quantifying DNA, RNA and proteins in both vitro and vivo. There
are a lot of different methods that you can use and upon the initial reading I was struggling to
understand how the tests correlated with detecting either DNA, RNA, or proteins and how
could you know if it was in vitro or vivo. For example I am able to distinguish that blotting is
a type of test for DNA, RNA, or proteins in vitro. However, at the time it is difficult for me
to remember that Southern blotting is a method for distinguishing DNA fragments, Northern
blotting tests for RNA, and Western blotting detects a particular protein (Griffiths et al.,
2020). I learned that the Fluorescence in Situ hybridization or FISH, the normal in situ
hybridization and immunofluorescence are used for distinguishing DNA, RNA, and proteins
in vivo (Griffiths et al., 2020). ISH is a method that uses hybridization with radioactive
single-stranded nucleic acid proteins to detect RNA or DNA (Griffiths et al., 2020). The
FISH method uses fluorescently labeled probes to detect DNA, and immunofluorescence is a
method that uses antibodies that are linked to fluorescent dyes to detect proteins in vivo
(Griffiths et al., 2020). The textbook also discussed the polymerase chain reaction test that is
used to detect DNA and a reverse transcription- PCR which is a method used to distinguish
RNA (Griffiths et al., 2020). Overall the methods are easy to understand when you can look
at how the tests are run and the actions that are taking place but if you’re trying to
memorize each method it can be much more difficult. This is why I plan to make flashcards
with the different tests on them so I can study and learn them which is better than just trying
to memorize what each test is used for.

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