Analytic Techniques

Question 1

Define:

chromatography

Answer 1

It 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.

Question 2

Name the two phases involved in any chromatography procedure.

Answer 2

1. stationary phase
2. 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.

Question 3

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

Answer 3

Column chromatography

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

Question 4

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

Answer 4

eluted from the column

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

Question 5

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.

Answer 5

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.

Question 6

Which type of column chromatography utilizes porous beads?

Answer 6

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.

Question 7

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?

Answer 7

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.

Question 8

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

Answer 8

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.

Question 9

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

Answer 9

Ion-exchange chromatography

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

Question 10

The diagram below depicts what type of chromatography?

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

Answer 10

Cation-exchange

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.

Question 11

The diagram below depicts what type of chromatography?

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

Answer 11

Anion-exchange

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.

Question 12

True or false:

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

Answer 12

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.

Question 13

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?

Answer 13

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.

Question 14

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

Answer 14

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.

Question 15

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

Answer 15

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.

Question 16

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?

Answer 16

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.

Question 17

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

Answer 17

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.

Question 18

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

Answer 18

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.

Question 19

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.

Answer 19

nonpolar, polar

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.

Question 20

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.

Answer 20

polar, nonpolar

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

Question 21

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

Answer 21

Ultraviolet-visible spectroscopy

(UV-Vis)

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.

Question 22

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

• Threonine
• Asparagine
• Tyrosine

Answer 22

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.

Question 23

Define:

gel electrophoresis

Answer 23

It 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.

Question 24

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

Answer 24

• Agarose (most common)
• Polyacrylamide
• Starch

Question 25

True or false:

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

Answer 25

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.

Question 26

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.

Answer 26

slower

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.

Question 27

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.

Answer 27

large

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.)

Question 28

Gel electrophoresis most closely resembles which type of electrochemical cell?

Answer 28

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.

Question 29

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

Answer 29

anode

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

Question 30

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.

Answer 30

smaller, larger

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

Question 31

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

Answer 31

anode

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

Question 32

True or false:

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

Answer 32

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.

Question 33

True or false:

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

Answer 33

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.

Question 34

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

Answer 34

bands

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

Question 35

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

Answer 35

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.

Question 36

Fill in the blanks.

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

Answer 36

homozygous, heterozygous

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.

Question 37

Fill in the blank.

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

Answer 37

size

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

Question 38

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.

Answer 38

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.

Question 39

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?

Answer 39

• 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.

Question 40

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?

Answer 40

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.

Question 41

Name the molecule shown here.

Answer 41

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.

Question 42

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

Answer 42

1. uniformly coats the proteins with a negative charge
2. 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.

Question 43

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

Answer 43

SDS-PAGE

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

Question 44

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

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

Answer 44

primary

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.

Question 45

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

Answer 45

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.

Question 46

Describe the four main steps of SDS-PAGE.

Answer 46

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.

Question 47

True or false:

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

Answer 47

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.

Question 48

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

Answer 48

blotting techniques

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

Question 49

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.

Answer 49

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.

Question 50

Fill in the blank.

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

Answer 50

northern

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

Question 51

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

Answer 51

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.

Question 52

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

Answer 52

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.

Question 53

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

Answer 53

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.

Question 54

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

Answer 54

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.

Question 55

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

Answer 55

ddNTPs

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.)

Question 56

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

Answer 56

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.

Question 57

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

Answer 57

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!

Question 58

What occurs in the annealing step of the PCR cycle?

Answer 58

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).

Question 59

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

Answer 59

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.

Question 60

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

Answer 60

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.

Question 61

Name three potential uses of recombinant DNA technology.

Answer 61

1. creating large amounts of purified DNA for further testing
2. amplifying specific sequences for use in gene therapy
3. producing recombinant proteins for use in medications

Question 62

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

Answer 62

DNA cloning or gene cloning

Question 63

Define:

a vector (in the context of DNA cloning)

Answer 63

It 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.

Question 64

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?

Answer 64

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.

Question 65

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:

Answer 65

a plate containing tetracycline

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

Question 66

The specific DNA sequences recognized by restriction enzymes are termed:

Answer 66

restriction sites or recognition sites

These sequences are generally 4-8 nucleotides in length.

Question 67

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

Answer 67

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.

Question 68

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

Answer 68

It 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.

Question 69

True or false:

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

Answer 69

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.

Question 70

True or false:

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

Answer 70

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.

Question 71

What does cDNA stand for?

Answer 71

complementary DNA

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

Question 72

What does gDNA stand for?

Answer 72

genomic DNA

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

Question 73

Unlike gDNA, cDNA does not contain:

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

Answer 73

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.

Question 74

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:

Answer 74

a DNA library

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

Question 75

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

Answer 75

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.

Question 76

Why does incorporation of a ddNTP terminate DNA replication?

Answer 76

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.

Question 77

True or false:

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

Answer 77

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.

Question 78

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

Answer 78

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.

Question 79

Name at least three practical applications of DNA technology.

Answer 79

1. Medical uses
2. Gene therapy
3. Pharmaceuticals
4. Forensic evidence
5. Environmental cleanup
6. Agriculture

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

Question 80

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.

Answer 80

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.