Blueprint Diagnostic Bio/BioChem Passage 4

Session 280

Today, we dissect passage 4 from the Biology/Biochemistry section of the Blueprint diagnostic exam.

We’re joined by George from Blueprint MCAT. If you would like to follow along on YouTube, go to premed.tv.

Listen to this podcast episode with the player above, or keep reading for the highlights and takeaway points.

[01:20] Tips to Avoid MCAT Prep Burnout

George explains that practice is the best place to make mistakes because it doesn’t matter. No one’s going to look at it. There are times throughout your prep where maybe it feels like concepts aren’t sticking, or maybe it’s just discouraging. You’ve put in a lot of work.

Just remember that each full-length is not going to cover the entire MCAT. They’re only going to cover a small section on full-length and another small section on your next one and another one.

It’s not until the later ones that you start seeing things over and over that things start to overlap. Many students in their prep, see the largest score increases at the tail end of their prep, which is hard to think about because you want to see progress right away.

It is easy to decide to get discouraged, that’s why it’s so important to reach out and get a good social network. Plan your breaks to make sure you’re disciplined with your study and your time off.

'Your mental health is just as important as your study health. They go hand in hand.'Click To Tweet

Prioritize your sleep, what you eat, your physical and mental health, and your social health as well. There are some things that you have to prioritize in the sense that you won’t go out five times a week, but still plan those things that you can look forward to.

This will keep you going. It will keep you fresh and motivated to study for the MCAT versus burying your head in a book for eight hours every day. Then you lose track of time, you don’t know what’s going on, and you’re not seeing your progress. It’s just a downward spiral. And so, find ways to lift yourself up when those things are down so you can keep yourself going.

[05:01] Passage 4 (Questions 19 – 23)

Paragraph 3

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation assays (PLA) and immuno-PCR. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. An important step in the synthesis of these molecules is sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions as shown in Figure 1.

Figure 1 Illustration of purification of affinity binder-oligonucleotide conjugates

Notes:

A lot of this stuff is presented as background. You probably don’t need to know all of it immediately. If it’s important, it will be expanded.

And so, high-performance methods are required for the isolation of pure conjugates, so you might highlight that. Ultimately, this comes back in that final sentence where it says removing any unconjugated affinity reagents and remaining freely illegal nucleotides.

This makes sense. We’re isolating something or trying to wash away the impurities. We want something specific and we’re probably going to see some methods.

If you look at Figure 1, you see an illustration of the purification process. All it says is an illustration of purification of affinity binder-oligonucleotide conjugates. Don’t spend too much time on it because if it’s important, they’ll explain it.

[07:14] Paragraph 2

First, the conjugation is performed in solution by mixing antibodies with an excess of azide-modified oligonucleotides. The resulting mix containing antibody-DNA conjugates and remaining free antibodies and oligonucleotides is incubated, and the products are immobilized on agarose beads. After washes to remove unconjugated antibodies, the captured antibody-DNA conjugates along with unconjugated oligonucleotides are both released by enzymatic cleavage using the restriction enzyme MlyI that cleaves at the border of the complementary region (Figure 2) between the capture and conjugate oligonucleotides to release single-stranded DNA-coupled conjugates. The cleavage process required both ATP and S-adenosyl-L-met in order to run to completion. The eluate was then captured on protein G beads and washes were conducted to remove any free oligonucleotides having no antibodies attached. Finally, purified conjugates were eluted from the protein G beads by lowering the pH.

Figure 2 Enzymatic cleavage by MlyI

Notes:

First, highlight conjugation, antibodies, and oligonucleotides. In my first stage, I’m going to take my strands of DNA and I’m going to conjugate them with something. In the next process, there’s going to be something that happens then they’re immobilized and washed.

The function is we’re removing something. We’re washing or getting rid of something right after because it says remove unconjugated antibodies. And so, just keep those things at the back of your mind.

What happens next is there’s a step of enzymatic cleavage, which you can highlight too. Again, orient yourself with the stages you recognize. Get rid of the fluff. It could be ABCD, but just try to orient yourself with the steps.

Then they remove free oligonucleotides and what you have left are pure conjugates. Then you have the final step of lowering the pH.

Again, just focus on just the steps. Don’t worry too much about what’s happening in the middle. If somehow you need to relate it, you can come back to the figure and paragraph so you can tie it all together.

[11:12] Paragraph 4

These purified high-grade probes extend the capabilities of traditional immunoassays to allow direct detection of proteins and modifications with high specificity and sensitivity. With them, designated protein targets can be readily detected and localized with single-molecule resolution and objectively quantified in unmodified cells and tissues.

Notes:

The author is just saying the process is great and we ultimately get what we’re looking for which is purified high-grade probes. The point here is they’re saying this method is great. And so, they described all the methods and the author says the method is great.

With that in mind, we’re ready to approach the questions and maybe come back to see if there’s anything we need from the passage.

[12:50] Question 19

In vitro, free oligonucleotides, such as mRNA transcripts, have a short half-life in the cytosol. What mechanism is used to stabilize these molecules?

A.Polyadenylation

B.Phosphorylation

C.Enzymatic digestion

D.Active transport across the nuclear membrane

Thought Process:

This is an example of a question where you don’t need any other information from the passage because you can answer it from your stored knowledge. It’s a pseudo-discrete question.

This is why you shouldn’t be fazed by these big ugly passages. They will throw it at you specifically to throw you off your game.

'There are tons of questions like this where they give you a tough passage and if it's a pseudo discrete and you know the content, you can answer the question.”Click To Tweet

Rephrasing the question, it’s about how to stabilize oligonucleotides such as mRNA, meaning what prevents degradation? The first step is figuring out what you need to answer.

C – With enzymatic digestion, you’re breaking it down so it’s not stabilizing. Hence, this is incorrect.

D – You want it in the cytosol so why would you transport it? This doesn’t make sense so we can throw this out too.

Now, we’re left with A and B.

The mRNA is the example that they gave us. When we think about transcription in ourselves, the biggest thing is we think of post-transcriptional modification. If we want to stabilize the transcript, we need to make modifications to this transcript to stabilize it. This is called a post-transcriptional modification.

Polyadenylation is a post-transcriptional modification that is slapped onto your mRNA to stabilize it. Poly means many, so you’re adding many.

Phosphorylation is involved in a lot of regulatory pathways for turning on and turning off proteins. In this case, we want to stabilize oligonucleotides so A is the best answer.

Correct Answer: A

[17:15] Question 20

Given the locations of the MlyI cleavage site and recognition sequence, MlyI is NOT a:

A.Type I restriction enzyme.

B.Type II restriction enzyme.

C.Type III restriction enzyme.

D.Type IV restriction enzyme.

Thought Process:

This is a very discreet content nugget. It was briefly mentioned in the passage that MlyI is a restriction enzyme. But this is such a specific thing where there really is no strategy. It’s like picking your favorite number.

There are just two things to keep in mind when you run across questions like this. Number one, never leave a question blank. And point number two, just guess and move on. Don’t give it extra time.

This is not a question you should kick yourself over because this is a very, very low-yield discrete question.

“This is one of those questions that you could stare at for five minutes, and you still have no extra information. So just pick your favorite number and move on.”Click To Tweet

That being said, George explains the thought process behind the correct answer. If we look at figure two, it shows that the cleavage site is super close to the recognition site.

Answer choice A is correct because we are looking for NOT here. Type I restriction enzymes are specific in the sense that they recognize a certain site, and they cut somewhere very far away.

If we look at figure two, we can see that just downstream or very close to our recognition site is our cut site. That does not satisfy our need for a Type I restriction enzyme. Therefore, MlyI is NOT a Type I restriction enzyme.

Type II restriction enzymes are considered anything that cleaves within or like short distances of your sequence site. So this would be fine since the cleavage site is very close.

Type III enzymes require S-Adenosyl methionine which was mentioned briefly in the passage,

Type IV enzymes are enzymes that target modified DNA, so methylated versus demethylated, or hydroxy, methylated.

We don’t really get any information for Figure 2 here. But you could reason out that if there’s a cytosine, you could slap on a methyl group or modify this DNA if that event was there.

Correct Answer: A

[21:52] Question 21

Which nucleophile was likely used to prepare the oligonucleotides used in the purification procedure?

A.NaOH

B.(CH3)3CCl

C.HCN

D.NaN3

Thought Process:

If you’re not expected to know it, this is one of those where you can probably find it in the passage.

Use the question to guide yourself about what a nucleophile is doing. Then look for where it’s being done in the steps that we laid out. In the passage, you can see azide-modified oligonucleotides. So they’re preparing the oligonucleotides by adding azide. So the key here is to look at which of the answer choices contains azide.

An azide is an ion where it’s an N3−. And looking at answer choice D, you see sodium with a plus ion and an N3. So it’s sodium azide.

Correct Answer: D

[27:35] Question 22

Some proteins and antibodies are susceptible to pH damage. In order to prevent this damage, what would be the most efficient and effective additional step to perform at the end of the elution process?

A.0.1 M glycine•HCl, pH 3.0

B.0.1 M NaCl, pH 7.0

C.1 M Tris•HCl, pH 8.5

D.0.7 M KOH, pH 13.85

Thought Process:

Going back to the steps, it says an additional step at the end of the elution process. And the immediate step before is they may make it more acidic. So you probably want to counteract that by adding something more basic.

The first part of the question says some proteins are very susceptible to pH damage. The most basic here is answer choice D. But again, remember they said the proteins are very susceptible to pH damage. If you want to prevent an overshoot, then you want to add a weak base. Therefore, C is the correct answer to prevent too much acidity while preventing the overshoot and using too much base to prevent damage as well.

Correct Answer: C

[33:38] Question 23

Which step of the procedure corresponds to the release of DNA-coupled conjugates?

A.B

B.C

C.E

D.D

Thought Process:

You’re looking in paragraph one for your answer here. Paragraph one says we have a process to purify some targets. And from start to finish, this is what we do, we remove the unconjugated affinity reagents, etc. The next paragraph is all the steps. The figure is a diagrammatic representation of the process. But then everything in this section here talks about that figure.

The question says released DNA coupled conjugates, so if you’re looking for cause and effect, something released these DNA coupled conjugates. And what happens before that is enzymatic cleavage.

In Step C, we’ve added this enzyme, and the little lines you see are the cleavage site where we’re cutting things. So you can notice that indeed, they’re a little bit shorter. You’re left with a region after you remove things. So D is the correct answer. D is the stage where the enzyme is added to kind of cleave things off.

Correct Answer: D

[40:27] Overcoming Self-Doubt

Remember, if you have that feeling of “I’m not smart enough” or you read a passage and you have no idea what’s going on, the point isn’t to know everything that’s going on in the passage.

The point is to remember that the MCAT tests the same core concepts, always. They will show you things to throw you off, just like we saw in this passage. They will give you really tough passages with a lot of information.

But take a step back and recognize that although you might not fully understand certain things, you’re going to try the questions anyway. And you’re going to get rewarded for it. That mindset is going to be rewarded so much more than having a mindset that you can’t do it.