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Flashcards in Analytic Techniques Deck (80)
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1
Q

Define:

chromatography

A

Chromatography is a set of techniques used to separate mixtures by passing them through a medium in which different components travel at different speeds.

On the MCAT, chromatography is most commonly used to separate proteins in biochemistry, although it can also appear in an organic chemistry context.

2
Q

Name the two phases involved in any chromatography procedure.

A

The stationary phase and the mobile phase

Chromatography utilizes the idea that, if the stationary phase and mobile phase have different properties, some components of the mixture will adhere to the stationary phase, while others will travel with the mobile phase.

3
Q

In this subset of chromatography, a liquid mixture is passed through a vertical tube packed with a solid adsorbent, such as silica beads.

A

Column chromatography

A number of more specific column chromatography techniques also exist, such as size-exclusion and ion-exchange chromatography.

4
Q

Components of a mixture that pass through and exit a chromatography column are said to have done what?

A

These components have eluted from the column.

In other words, the term “elute” means “to pass through” in the context of a chromatography column.

5
Q

A biochemist finds that, while his column chromatography procedure is helping to purify his mixture, it has very poor resolution. What change can he make to the column to help address this?

Assume that the biochemist cannot change the identities of the stationary and mobile phases.

A

The biochemist can increase the length of the column.

In column chromatography, poor resolution means that different compounds are not eluting with enough separation to effectively purify the mixture. Lengthening the column greatly slows the process, but (as in many lab techniques) a slower process corresponds to a better result, which, here, is clearer separation.

6
Q

Which type of column chromatography utilizes porous beads?

A

Size-exclusion chromatography

Porous beads are simply beads with tiny holes in their structures. Size-exclusion chromatography takes advantage of the fact that smaller molecules will become trapped in these beads and adhere to the column.

7
Q

A mixture of alanine, tryptophan, and histidine is passed through a size-exclusion chromatography apparatus. Which of these amino acids is expected to elute most slowly?

A

Alanine

In size-exclusion chromatography, the smallest particles actually elute the most slowly, as they become trapped in the beads of the stationary phase.

Alanine, the second simplest amino acid, is significantly smaller than the other two options here.

8
Q

In the size-exclusion separation of proteins, retention time has what kind of relationship with the parameter measured in kilodaltons?

A

An inverse relationship

Kilodaltons (and daltons) are the units for protein molecular weight. Molecular weight generally corresponds to size, which is inversely related to the amount of time a molecule spends in a size-exclusion column.

9
Q

Which type of column chromatography exploits differences in the charges of the mixture’s components?

A

Ion-exchange chromatography

As its name indicates, ion-exchange chromatography separates proteins (or other components of a mixture) on the basis of charge.

10
Q

The diagram below depicts what type of chromatography?

Choose from anion-exchange, cation-exchange, or neither.

A

Cation-exchange chromatography

Since the stationary phase in this diagram is negatively-charged, this chromatography method would retain cations and allow anions to pass through the column. Therefore, this is cation exchange.

11
Q

The diagram below depicts what type of chromatography?

Choose from anion-exchange, cation-exchange, or neither.

A

Anion-exchange chromatography

Since the stationary phase in this diagram is positively-charged, this chromatography method would retain anions and allow cations to pass through the column. Therefore, this is anion exchange.

12
Q

True or false:

Ion-exchange chromatography techniques are named for the type of ion that elutes through the column most rapidly.

A

False

This is the reverse of the truth! In reality, ion-exchange techniques (specifically, anion- and cation-exchange chromatography) are named for the ion that adheres to the beads in the column.

13
Q

Two similarly-sized proteins pass through an anion-exchange column: a protein rich in valine and isoleucine and a protein rich in glutamate. Which protein will display a higher retention time?

A

The glutamate-rich protein

Glutamate is an amino acid that is negatively-charged (at least at physiological pH). In contrast, valine and isoleucine are neutral. In anion-exchange chromatography, negative species are retained on the column, leading to a higher retention time.

14
Q

Once cationic mixture components have adhered to a cation-exchange column, how can they be removed?

A

The column can be flushed with an even more positive solution.

This will displace the cationic mixture components from the column, allowing them to be collected for later use.

15
Q

Which type of column chromatography leads to by far the most specific separation?

A

Affinity chromatography

In this technique, the stationary phase is engineered to selectively bind the molecule of interest (often using antibodies for that molecule). This leads to far better separation than the broader, characteristic-based chromatography methods.

16
Q

One chromatography technique involves tagging a genetically modified protein of interest with histidine residues and then passing it, in a mixture, through a column designed to bind histidine. Which type of column chromatography is this?

A

Affinity chromatography

Since the column here is specifically designed to bind histidine, this technique exemplifies affinity chromatography, which is far more specific than size-exclusion or ion-exchange chromatography.

17
Q

An ion-exchange procedure involves the use of lysine-bound silica beads and a very low-pH mobile phase. Will this procedure be effective?

A

It will not be effective.

Specifically, the stationary phase and the mobile phase are too similar here, as both are highly positive (remember, low pH = high [H+]). Effective chromatography requires that the stationary phase and the mobile phase be different with regard to the relevant parameter.

18
Q

A form of chromatography in which the mixture is pushed through the column by extremely high pressures is termed:

A

high-performance liquid chromatography (HPLC).

Essentially, HPLC works like regular column chromatography, except the high pressures allow it to take place more quickly while still yielding very good resolution.

19
Q

Normal-phase HPLC has a [polar/nonpolar] mobile phase and a [polar/nonpolar] stationary phase.

Choose one term from each box above to accurately complete the sentence.

A

Normal-phase HPLC has a nonpolar mobile phase and a polar stationary phase.

One method that can help you remember this is to notice that it resembles typical thin-layer chromatography, which most of us are somewhat more familiar with.

20
Q

Reverse-phase HPLC has a [polar/nonpolar] mobile phase and a [polar/nonpolar] stationary phase.

Choose one term from each box above to accurately complete the sentence.

A

Reverse-phase HPLC has a polar mobile phase and a nonpolar stationary phase.

Interestingly, this HPLC method is used more frequently than normal-phase HPLC (and may be somewhat more likely to appear on the MCAT, too).

21
Q

A student finds that a particular protein strongly absorbs light with a wavelength of 280 nm. Which technique was this student most likely using?

A

Ultraviolet-visible (UV-Vis) spectroscopy

Since visible light has a wavelength of 400-700 nm, it is logical that 280 nm would fall in the ultraviolet (essentially, “higher frequency/lower wavelength than violet visible light”) range. Ultraviolet light is utilized by UV-Vis spectroscopy.

22
Q

Which of the following amino acids is likely to display a strong absorbance at 280 nm in UV-Vis spectroscopy?

  • Threonine
  • Asparagine
  • Tyrosine
A

Tyrosine

If you know nothing else about UV-Vis spec, you should understand that it is commonly used to identify the presence of conjugated or aromatic species. Tyrosine is the only aromatic amino acid listed.

23
Q

Define:

gel electrophoresis

A

Gel electrophoresis is a separatory technique in which a mixture is moved through a gel by an electric current.

Depending on their characteristics, the mixture components travel at varying rates down the gel, causing them to be present at varying positions at the end of the procedure.

The goal of such a procedure is typically to separate molecules by size.

24
Q

Name at least one substance that commonly makes up the gel in gel electrophoresis.

A

You could have named any of the following substances:

  • Agarose (most common)
  • Polyacrylamide
  • Starch
25
Q

True or false:

In gel electrophoresis, smaller molecules travel more quickly and move farther down the gel than larger molecules.

A

True

Smaller molecules are better able to move through the matrix (porous structure) of the gel. In contrast, large molecules encounter more resistance and travel more slowly and less far.

26
Q

A gel created with a higher percentage of agarose will lead to [slower/faster] movement of the mixture components through it.

Choose one term from the box above to accurately complete the sentence.

A

A gel created with a higher percentage of agarose will lead to slower movement of the mixture components through it.

A higher percentage of agarose (as opposed to water, which is the other main component of an agarose gel) will cause the gel to be thicker and more solid. Substances will travel relatively slowly through such a gel.

27
Q

A gel created with a lower percentage of agarose would be more effective at resolving [small/large] molecules.

Choose one term from the box above to accurately complete the sentence.

A

A gel created with a lower percentage of agarose would be more effective at resolving large molecules.

Molecules will travel relatively quickly through this thin gel. Therefore, such a gel is best for resolving large molecules. (In contrast, if a thick, agarose-rich gel were used to resolve large molecules, all of those molecules would likely move so slowly that they would be difficult to distinguish from each other.)

28
Q

Gel electrophoresis most closely resembles which type of electrochemical cell?

A

An electrolytic cell

Electrolytic cells, like gel electrophoresis, must be powered by an outside power source to overcome the nonspontaneity of the process.

Isoelectric focusing, discussed in the Amino Acid Properties deck, similarly resembles an electrolytic cell.

29
Q

In gel electrophoresis, would negatively-charged molecules be drawn toward the cathode or the anode?

A

Negatively-charged molecules would be drawn toward the anode.

As in an electrolytic cell, the anode of a gel electrophoresis apparatus is positively-charged.

30
Q

In gel electrophoresis, [smaller/larger] molecules travel more rapidly.

In size-exclusion chromatography, [smaller/larger] molecules travel more rapidly.

Choose one term from each box above to accurately complete the sentences.

A

In gel electrophoresis, smaller molecules travel more rapidly.

In size-exclusion chromatography, larger molecules travel more rapidly.

Make sure you remember this! In particular, size-exclusion chromatography is important to recall, as it can seem counterintuitive.

31
Q

In the electrophoresis of DNA, the DNA molecules move toward the:

A

anode.

Due to its highly negative phosphate groups, DNA is negatively-charged overall! Therefore, it is drawn toward the positive anode.

32
Q

True or false:

In gel electrophoresis, DNA is initially loaded into wells at the cathode side of the gel.

A

True

Since DNA moves toward the anode during the electrophoretic procedure, it is initially loaded onto the gel near the cathode end. That way, it can move down the length of the gel in response to the applied current.

33
Q

True or false:

In gel electrophoresis, DNA can be separated with little or no pre-treatment of the DNA-containing sample.

A

True

Since DNA is already negative and contains negative charges in proportion to its mass, it will travel down the gel in a manner that is dependent only on its size (which is the purpose of gel electrophoresis). It does not require pre-treatment with any reagents.

34
Q

The lines on a gel that correspond to DNA fragments of different sizes are termed:

A

bands.

For example, a band might include fragments that are 500 base pairs (bp) long.

35
Q

What pre-determined mixture is often run alongside DNA samples to allow for easy comparison of the sizes of different bands?

A

A molecular weight marker or DNA ladder

Regardless of which of the above terms is used, the takeaway is that this provides an easy standard of comparison using one lane of the gel.

This resembles a positive control, as essentially, we are running a substance with known results (for example, bands of 500 bp, 1000 bp, etc.) alongside our sample.

36
Q

With regard to genotyping, a ________ genotype will be reflected by a single band on gel electrophoresis, whereas the ________ genotype typically appears as two bands.

A

With regard to genotyping, a homozygous genotype will be reflected by a single band on gel electrophoresis, whereas the heterozygous genotype typically appears as two bands.

While you should still pay close attention to the described technique if it appears in a question or passage, this rule of thumb can be helpful.

37
Q

DNA electrophoresis is particularly useful for separating DNA fragments by ________ after a restriction digest.

A

DNA electrophoresis is particularly useful for separating DNA fragments by size after a restriction digest.

“Length” (in base pairs) would also work as a correct answer here.

38
Q

True or false:

A DNA molecule is cut into three fragments in a restriction digest. Therefore, three distinct bands must appear on an electrophoresis gel.

A

False

While this may occur, it won’t necessarily occur! In particular, if two or more of the fragments are the same length, their bands will “merge,” leading to the presence of fewer than three distinct bands.

39
Q

Name two reasons why straightforward gel electrophoresis does not work with proteins.

In other words, why does it not separate proteins on the basis of size alone?

A
  • Proteins (unlike DNA) possess higher-level folding structure, leading to undesired variations in movement through the gel.
  • Proteins (unlike DNA) do not all possess a uniform charge; in fact, some may not be charged at all.
40
Q

Two proteins of similar size and with similar high-level folding are passed through a simple gel electrophoresis procedure. One protein has a pI of 4.7, while the other has a pI of 9.0. Which one will travel farther down the gel?

A

The protein with the pI of 4.7

A lower pI corresponds to a greater prevalence of negatively-charged residues. This will cause the protein to travel farther toward the positively-charged anode than the higher-pI protein.

41
Q

Name the molecule shown here.

A

This molecule is sodium dodecyl sulfate (SDS)

The “sodium” in this name refers to the Na+ ion shown, the “dodecyl” to the 12-carbon chain, and the “sulfate” to the negatively-charged sulfate head of the molecule.

42
Q

Name two functions that SDS accomplishes with regard to the separation by size of proteins.

A

SDS:

  • uniformly coats the proteins with a negative charge
  • denatures higher-level structure

This second function is accomplished because SDS is a detergent, or amphipathic molecule able to denature (disrupt) many interactions involved in high-level folding.

43
Q

A special type of electrophoresis that is used for proteins and that utilizes SDS is:

A

SDS-PAGE.

This abbreviation stands for “sodium dodecyl sulfate polyacrylamide gel electrophoresis.”

44
Q

While largely effective at denaturing proteins, SDS does not disrupt ________ protein structure at all.

Choose from primary, secondary, tertiary, or quaternary structure.

A

While largely effective at denaturing proteins, SDS does not disrupt primary protein structure at all.

Denaturation in general does not impact primary structure! In this example in particular, we also wouldn’t want to disrupt the primary structure, as that would break apart the protein, rendering future separation/analysis pointless.

45
Q

Name the only covalent bonds found in higher-level protein structure, which are not impacted by treatment with SDS.

A

Disulfide bonds

Since disulfide bonds are covalent, they are not denatured by SDS, a detergent that disrupts intermolecular attractions. For this reason, proteins are also often treated with a reducing agent prior to electrophoresis, as such a reagent breaks S-S bonds.

46
Q

Describe the four main steps of SDS-PAGE.

A
  1. Treat the sample(s) with SDS (and, often, with a reducing agent).
  2. Load the sample(s) vertically onto a polyacrylamide gel.
  3. Turn on the electric current.
  4. Turn off the current, and stain the gel with a reagent that allows for visualization of the bands.
47
Q

True or false:

The SDS-PAGE separation of a purified sample containing a dimeric protein will always produce two distinct bands on the gel.

A

False

This may be tempting, as SDS denatures quaternary structure, which separates protein subunits! However, this statement is only accurate if the two subunits are different sizes. If they are the same size (as in a homodimer), they will travel the same distance and will be seen as a single band on the gel.

48
Q

Techniques in which proteins, RNA, or DNA are transferred onto a membrane for subsequent labeling and/or visualization are termed:

A

blotting techniques.

A number of these techniques exist, most notably Southern, northern, and western blotting.

49
Q

Which type of blotting is used to detect specific DNA sequences?

Hint: This was the first type of blotting developed and is the only type to be directly named after a person.

A

Southern blotting

Named for Edwin Southern, this technique is used to detect DNA sequences. As the other forms of blotting are used for other types of molecules (namely proteins and RNA), a mention of DNA should be enough for you to recognize Southern blotting.

Interestingly, the other blotting techniques (such as western blotting) are not named for people, but rather were named to continue the trend of direction-related terms.

50
Q

The detection of specific ribonucleic acid sequences can be accomplished with ________ blotting.

A

The detection of specific ribonucleic acid sequences can be accomplished with northern blotting.

Ribonucleic acid (or RNA) is the focus of northern blotting. In contrast, Southern blotting is used for DNA sequences.

51
Q

This form of blotting is often preceded by the electrophoresis of the protein-containing sample.

A

Western blotting

The mention of proteins here should be enough to tell you that we are dealing with western blotting! In contrast, Southern blots are used for DNA, while northern blots are utilized for RNA.

52
Q

A neurogeneticist is looking to identify the sequence 5′-ACUUGAUGGCCU-3′ in a sample. Which form of blotting should she use?

A

Northern blotting

The inclusion of uracil (U) in this sequence tells us that it must be a sequence of RNA. Northern blotting is used for the detection of specific RNA sequences.

53
Q

Name the three main steps of a polymerase chain reaction (PCR).

A

The three steps of PCR are as follows:

  1. Denaturation of the template dsDNA
  2. Annealing of the primers
  3. Extension of the new DNA strands

Essentially, PCR can be thought of as DNA replication conducted in a lab.

54
Q

A polymerase chain reaction (PCR) mixture typically contains a number of reagents. Among these, name the enzyme that is usually used.

A

Taq polymerase

Isolated from a thermophilic bacterium, Taq polymerase is a heat-stable DNA polymerase. It is commonly used in PCR procedures due to its ability to withstand the temperature fluctuations involved without becoming denatured.

55
Q

Which of the following is NOT a reagent used in a typical PCR procedure?

  • DNA buffer
  • Mg2+ ions (or a source of these ions, such as MgCl2)
  • Primers
  • ddNTPs
A

ddNTPs are not a reagent used in PCR.

ddNTPs stands for dideoxynucleotide triphosphates. These nucleotides are used in Sanger sequencing, not in PCR. (PCR utilizes dNTPs, which possess the 3′ OH group needed to facilitate continued extension of the DNA strand.)

56
Q

What is the role of Mg2+ (often included in the reaction mixture as MgCl2) in PCR?

A

Mg2+ acts as a cofactor for Taq polymerase.

Many metal cations serve as cofactors for enzymatic reactions! Mg2+ in particular is often found as a cofactor for reactions that involve DNA, in large part because its positive charge can stabilize the highly negative DNA backbone.

57
Q

Which of the three steps of the PCR cycle takes place at the highest temperature?

A

Denaturation

Since denaturation involves separating (or denaturing) the double-stranded DNA template, it must occur at a relatively high temperature (94-98 °C). Remember, heat is a powerful denaturant!

58
Q

What occurs in the annealing step of the PCR cycle?

A

In response to lowered temperature, complementary primers anneal to (or hybridize with) the now single-stranded template DNA molecules.

The temperature during this step is 50-65 °C, which is much lower than that of the prior step (denaturation).

59
Q

Taq polymerase is most catalytically active during this step of the PCR cycle.

A

Extension

During this step, Taq polymerase catalyzes the extension of the new DNA strands complementary to the template strands. This step takes place at a temperature between those of the previous two steps.

60
Q

What is the formula used to calculate the number of DNA copies produced after a certain number of PCR cycles?

A

2n

Here, the variable n corresponds to the number of cycles that have taken place. The “2” stems from the fact that the number of copies produced doubles every step.

61
Q

Name three potential uses of recombinant DNA technology.

A

Recombinant DNA technology has a wide array of purposes, including:

  • creating large amounts of purified DNA for further testing
  • amplifying specific sequences for use in gene therapy
  • producing recombinant proteins for use in medications
62
Q

The synthesis of multiple identical copies of a particular DNA sequence of interest is termed:

A

DNA cloning or gene cloning.

63
Q

Define:

a vector (in the context of DNA cloning)

A

A vector is a molecule of DNA that is used to bring foreign (often recombinant) genetic material into a cell.

On the MCAT, vectors are most commonly plasmids, which are small, circular dsDNA molecules often found in bacteria.

64
Q

What concept is often utilized in DNA recombination to ensure that the recombinant plasmid actually contains the DNA fragment of interest and that the cells being used have actually picked up the plasmid?

A

Antibiotic resistance

Essentially, a sequence that codes for antibiotic resistance is included in the fragment of interest. If recombination works properly and if the cells pick up the plasmid, they will be resistant to the antibiotic. If the process fails, the antibiotic will kill the cells.

65
Q

A recombinant plasmid includes a small fragment coding for tetracycline resistance. To test whether recombination and transformation worked properly, scientists should next grow the transformed cells on:

A

a plate containing tetracycline.

This is common practice during DNA recombination procedures, although the specific antibiotic used can vary.

66
Q

The specific DNA sequences recognized by restriction enzymes are termed:

A

restriction sites or recognition sites.

These sequences are generally 4-8 nucleotides in length.

67
Q

Restriction enzymes often create 3’ or 5’ overhangs. Name the jagged ends of DNA strands cleaved with these enzymes.

A

Sticky ends

Sticky ends are particularly useful because they allow DNA fragments cut with the same restriction enzyme to “fit into” each other complementarily, which is not the case with blunt ends.

68
Q

In the context of double-stranded DNA, what is a palindromic sequence?

A

A palindromic sequence is a sequence in which one strand, read 5′ to 3′, has the same sequence as its complementary strand read 3′ to 5′.

The recognition sites for restriction enzymes are typically short palindromic sequences.

69
Q

True or false:

A double-stranded DNA sequence that reads AAGCTT along one strand is a palindromic sequence.

A

True

In the context of DNA and restriction enzymes, palindromes are sequences where one strand (read 5′ to 3′) is identical to the sequence of the complementary strand read 3′ to 5′. Here, the complementary strand would be TTCGAA, so reading it in the opposite direction as the given strand would produce the same sequence, making this a palindrome.

In fact, the provided sequence is the restriction site for the enzyme HindIII.

70
Q

True or false:

A double-stranded DNA sequence that reads GATTAG along one strand is a palindromic sequence.

A

False

Remember, to assess whether a DNA sequence is palindromic, you must consider both complementary strands (not just one!). Here, the complementary strand is CTAATC, which reads entirely differently from either direction than GATTAG.

71
Q

What does cDNA stand for?

A

complementary DNA

cDNA is generated from a single-stranded RNA template by the enzyme reverse transcriptase.

72
Q

What does gDNA stand for?

A

genomic DNA

gDNA is regular, chromosomal DNA. An organism’s complete set of genomic DNA is termed its genome.

73
Q

Unlike gDNA, cDNA does not contain:

Hint: Consider how cDNA is generated, particularly the template used for that process.

A

introns.

This is the most important difference between gDNA and cDNA! Since cDNA is synthesized from an RNA template using reverse transcriptase, and mature RNA lacks introns, cDNA lacks introns as well.

74
Q

A collection (often large) of DNA fragments that have been isolated, cloned into vectors, and transformed into micro-organisms for storage and later analysis is termed:

A

a DNA library.

Multiple forms of DNA libraries exist, most notably complementary DNA (cDNA) and genomic DNA (gDNA) libraries.

75
Q

What technique is used to determine the nucleotide order in a DNA molecule via the incorporation of dideoxynucleotides?

A

Sanger sequencing

Dideoxynucleotides, or ddNTPs, are nucleotides that terminate the elongation of a DNA strand. Since many DNA molecules exist in solution, they experience termination at different points in their replicating strands, and (usually via labeling of the ddNTPs) this facilitates the identification of the nucleotide at each position.

76
Q

Why does incorporation of a ddNTP terminate DNA replication?

A

A ddNTP lacks a 3′ -OH group, which is required for the addition of the subsequent nucleotide in DNA replication.

DNA chain elongation requires a 3′ -OH group to “build off of,” which is actually why primers are required in in vivo DNA replication. With no 3′ -OH, replication will halt.

77
Q

True or false:

In Sanger sequencing, the concentration of ddNTPs must be far greater than the concentration of regular dNTPs.

A

False

While this statement is accurate in that both ddNTPs and regular dNTPs are used in Sanger sequencing, it reverses the relative concentrations. If [ddNTPs] was far greater than [dNTPs], the vast majority of replication reactions would terminate after the first or second nucleotide, and sequencing the entire strand would be impossible.

78
Q

What type of cell is used extensively in biotechnology, most commonly in the study of regenerative medicine?

A

Stem cells

These are cells with remarkable developmental ability, in that they can give rise to a variety of different cell types. Stem cells are currently being researched in a variety of regenerative contexts.

79
Q

Name at least three practical applications of DNA technology.

A

Applications of DNA technology include:

  • Medical uses
  • Gene therapy
  • Pharmaceuticals
  • Forensic evidence
  • Environmental cleanup
  • Agriculture

While others certainly exist as well, the list above is what the AAMC specifies on the official MCAT outline.

80
Q

Perhaps the most famous case involving ethics (or the lack thereof) in DNA research and technology involved a patient named:

Hint: This patient’s cancer cells were taken without her knowledge and used to culture a cell line still used today.

A

Henrietta Lacks

This case highlighted the need for consent, privacy, and patient rights.

While this foray into medical history may seem random, note that the AAMC does specifically list the safety and ethics of DNA technology on its official outline.