INTRODUCTION

General Introduction

A key process in cancer diagnostics is identifying mutations in tumor suppressor

genes and oncogenes. In 2014, Dr. Carl Wittwer stated that “a revolution [is] in

progress” regarding molecular diagnostics, adding that “The number of targeted cancer

therapeutics continues to increase” (Chiu et al., 2015). In this thesis, I will explore the

use of a finicky molecular beacon probe called KRAS G12V to identify a particular

single-nucleotide polymorphism (hereafter referred to as a “SNP”) in the oncogene KRAS

under two hypotheses: (1) It is possible to develop an assay using linear-after-the-

exponential-PCR (LATE-PCR) and the aforementioned molecular beacon probe to

distinguish the KRAS G12V SNP from other KRAS genotypes and (2) said LATE-PCR

assay can be developed through design of new primers. The G12V SNP is a guanine to

thymine mutation, so named because the wild-type gene codes for glycine and the

mutation produces valine (Chan, 2014). It is one of six potential cancer-related SNPs that

can occur in codon twelve of KRAS (Chan, 2014). Sequences can be found on page 58 of

the appendix.

In order to fully explicate this project, I will describe the use of real-time

quantitative polymerase chain reactions (hereafter referred to as RT-qPCR) and LATE-

PCR and the optimization of both of those PCR formats. I will detail the design and

mechanism of the KRAS G12V molecular beacon probe, the implementation of the assay

employing the probe with LATE-PCR, and the results of that assay probe. I will

conclude by discussing the further implications of my work on the use of molecular

beacons designed to detect SNPs in the KRAS gene.

Identifying which of a potentially large number of SNPs may or may not be

present in a given gene is immensely advantageous. Current technology relies on PCR in

conjunction with fluorescent probes such as molecular beacon probes, TaqMan probes,

and adjacent hybridization probe assays (Kwok et al., 2003). While there are multiple

methods of SNP detection, there are advantages to using molecular beacons for this

purpose. For example, TaqMan probes rely on the activity of Taq DNA polymerase,

limiting the experimenter to PCR assays that can only be performed using extension

phase temperatures at which Taq activity is optimal (Shiotani et al., 2014). Conversely,

molecular beacons can be designed after the PCR is optimized, allowing the experimenter

greater flexibility (Tyagi & Kramer, 1996).

Real-Time Quantitative Polymerase Chain Reactions: The Basics

PCR has been a useful technique in biotechnology since before the use of the

thermostable Thermus aquaticus ("Taq") polymerase in PCR reactions, prior to which

new polymerase molecules had to be added to each sample at each new step and thermal

cyclers had to be designed to allow for this (Henson, 2004). In 1985, Taq polymerase

was successfully synthesized in vitro; in 1991, the first rapid thermal cycler for PCR was

patented and put into production (Henson, 2004). This advance allowed PCR to become

considerably more useful (Lo, 2014). The rest is history, but it is a history that is still

ongoing, so it might be more accurate to refer to this “history” as a present and a future as

well.

The polymerase chain reaction is a delicate assay that depends on a number of

reagents: the aforementioned thermostable polymerase, two short (ordinarily 10-20

nucleotides) single-stranded oligonucleotide sequences – called primers – complementary

to the 3’ end of both the sense and antisense strands of the region to be amplified,

deoxyribonucleotide triphosphates (called dNTPs), magnesium or manganese cations,

and a buffer solution that is optimal for the polymerase of choice. Each individual

reaction consists of these components as well as DNAse-free water, DNA template, and –

in the case of RT-qPCR – a fluorescent probe or reagent. Under the proper conditions,

the primers hybridize to their target sequence on their complementary strands, the

polymerase binds at the 3’ end of the primer, and the polymerase extends the primer to

form a new strand complementary to the target strand using the dNTPs. Each new strand

is referred to as an amplicon (Brunstein, 2013).

One cycle of PCR includes three stages: denaturation, annealing of primers, and

extension of the primers. During the extension stage, new amplicons are created

(Brunstein, 2013). At 100% efficiency, each target strand is replicated during one cycle,

causing an exponential increase in the number of amplicons with each cycle. The

number of cycles in a PCR assay may range from 35 to 80 depending on the amount of

target DNA in the initial sample and the amount of product required for detection. Figure

1.1 depicts the accumulation of DNA product within the first four cycles assuming 100%

efficiency (Hollis, 2013).

Figure 1.1: Visual representation of the exponential increase in DNA strands during four cycles of PCR. Modified image from Hollis, 2013.

A major difference between traditional PCR and RT-qPCR is that detection of the

resulting amplicons is only possible with traditional PCR following the reaction’s

completion, while RT-qPCR allows detection while the reaction is ongoing. Another

major difference between the two formats of PCR is that RT-qPCR is quantitative

(Applied Biosystems, 2014). Another advantage of RT-qPCR is that tubes are always

closed for such an assay, avoiding cross-contamination. RT-qPCR also allows the

experimenter to control at which stage of the PCR the fluorescence signal is detected by

the spectrofluorometric thermal cycler. In the case of RT-qPCR using SYBR Green, the

SYBR Green is not fluorescent until it intercalates into double-stranded DNA (dsDNA)

(Deprez et al., 2002). This is demonstrated in Figure 1.2. It is also important to mention

that SYBR Green fluorescing only when intercalated into dsDNA is not an absolute; it is

normal for SYBR Green to produce a lower level of fluorescence in the presence of

single-stranded DNA (ssDNA). As such, SYBR Green emits a fluorescent signal

detectable by the appropriate PCR instrument upon excitation by a laser during the

annealing and extension stages (Deprez et al., 2002). The results from the quantitation of

fluorescence at each annealing and/or extension stage can be graphed, visualizing the

accumulation of the amplicons (Gevertz et al., 2005).

Figure 1.2: Depiction of how SYBR Green intercalates into double-stranded DNA, producing fluorescence during the annealing and extension stage of PCR. Modified image from Zipper et al., 2004.

At a certain point in the PCR, the signal is intense enough to surpass the

background fluorescence and become detectable by the instrument. This point is

quantified by a value called the threshold cycle (CT) (Gevertz et al., 2005). This term

refers to the cycle at which the reaction switches over from the exponential stage to the

pseudolinear stage, i.e., crosses the threshold from an undetectable signal to a detectable

signal (Applied Biosystems, 2014). The CT value is heavily dependent on the initial

concentration of target DNA and is calculated by a standardized statistical method

(Gevertz, 2005). Figure 1.3 depicts an RT-qPCR amplification plot and threshold line of

a PCR assay carried out for this thesis.

Figure 1.3: Typical RT-qPCR amplification plot, with cycle as the abscissa and fluorescence intensity as the ordinate; a method of quantifying the accumulation of amplicons. The threshold line, in black, indicates the CT value for the listed PCR reactions.

Linear-after-the-Exponential-PCR

As mentioned previously, a major advantage of real-time quantitative PCR over

traditional PCR is that traditional PCR does not yield data about the CT (Applied

Biosystems, 2014). CT is dependent on the initial concentration of target DNA and the

efficiency of the PCR, and as such CT is a useful metric by which to gauge the efficiency

of the assay. PCR assay efficiency is dependent on a variety of factors, including primer

concentration and bivalent cation concentration (Pfaffl, 2001). When there is a

significant difference between the forward and reverse primer concentration for the

purpose of producing more of one of either the sense or antisense strand amplicons, the

protocol is referred to as asymmetric PCR (Sanchez et al., 2004). While this has the

advantage of producing more of the strand to which a fluorescent probe binds, it has the

disadvantage of frequently being 20-30% less efficient than symmetric PCR (Sanchez et

al., 2004).

For this thesis, an improved type of asymmetric PCR called linear-after-the-

exponential PCR (hereafter referred to as LATE-PCR) was used. This subset of PCR is

so called because it is designed to avoid the tendency of symmetric PCR to plateau

stochastically as a result of PCR products competing with primers for binding to the

DNA target; therefore, the de novo synthesis of PCR product can be linear, as opposed to

plateauing, after the exponential stage (Pierce et al., 2005). LATE-PCR is an ingenious

method of producing an excess of one product strand without sacrificing efficiency. The

reason non-LATE asymmetric PCR is so inefficient compared to its symmetric

counterpart is that the Tm of the limiting primer is dependent upon its concentration

(Owczarzy, 2015). When asymmetric PCR first was developed, its low efficiency posed

a seemingly incorrigible conundrum until it was realized that lowering the primer

concentration lowered the melting temperature of the limiting primer, decreasing

efficiency with which the primer binds to its template (Sanchez et al., 2004).

The concept of LATE-PCR is simple: the limiting primer is redesigned so that it

has a Tm that is higher than the Tm of the excess primer (Pierce et al., 2005). This

calculation can be done with software that also takes into account cation concentration,

another factor that affects melting temperature. The task of increasing melting

temperature is accomplished by increasing the length of the primer. For optimal

efficiency in LATE-PCR, the melting temperature (Tm) of the limiting primer should be

between 5 °C and 7 °C above the Tm of the excess primer (Sanchez et al., 2004).

Finicky Molecular Beacon Probes

LATE-PCR is ideal for use with molecular beacon probes due to the fact that each

molecular beacon probe is designed to bind only to the sense strand. As such, the best

results with a molecular beacon are obtained when the PCR produces an excess of the

sense strand (El-Hajj et al., 2009). This way, there are far fewer antisense strands present

to compete with the molecular beacon probe for binding.

Working with molecular beacons requires a significant understanding of their

underlying mechanism. While there exist many fluorescent probes that rely on

fluorescence detected when a probe’s fluorescent moiety and quencher are no longer in

close proximity due to the probe binding to its target (Kwok et al., 2003), molecular

beacon probes are unique in their structure. They are single-stranded oligonucleotides,

ordinarily about 30 nucleotides long, which form a hairpin structure at room temperature

in the absence of their target (Tyagi & Kramer, 1996). They have two “arm” sequences

that are complementary to each other and a loop sequence that is designed to be

complementary to the mutation to which the probe is designed to bind (Tyagi & Kramer,

1996).

A quencher molecule is linked to one end of the probe and a fluorescent moiety is

linked to the other end of the probe. Extensive experimentation by the probes’

developers has yielded a list of the optimal fluorophore(s) for use with each quencher

(Marras, 2008). Interestingly, when molecular beacon probes were invented, the

fluorescence signal-detection technique that the probes were designed to use was

fluorescence resonance energy transfer (FRET) (Tyagi et al., 1998). However, when the

probes were initially tested, the quencher first used (dabcyl), did not behave as expected –

in fact, it was capable of quenching fluorescence from fluorophores whose emission

spectra did not overlap with its absorption spectrum – leading the inventors to conclude

that FRET was not the mechanism of quenching. It was then discovered that when a

molecular beacon probe’s fluorophore and quencher are in close enough proximity,

chemical bonds are formed between the moieties that prevent photon emission by the

fluorophore (Marras et al., 2002).

When the probe has a hairpin structure (referred to as the “closed” state), the

quencher and the fluorescent moiety are in close enough proximity that the quencher

bonds to the fluorophore, preventing the fluorescent moiety from producing a signal

(Tyagi et al., 1998). The reason molecular beacon probes produce a fluorescent signal in

the presence of their target is that it is thermodynamically more favorable for the probe to

bind to its target than to itself (Marras, 2003). This is illustrated in Figure 1.4. This

configuration is referred to as the “open” state (Kramer et al., 2009).

Figure 1.4: Operation of molecular beacon probes displaying the lack of fluorescence in the “closed” configuration. When the fluorescent moiety and the quencher are held at a distance by the rigidity of the beacon/target hybrid’s double helix structure, the fluorescent moiety can emit photons. Modified from Tyagi & Kramer et al., 1996.

There are two types of molecular beacon probes: “sloppy” and “finicky”. As

demonstrated in Figure 1.5, sloppy molecular beacon probes are designed to be tolerant

of multiple mismatched base pairs (El-Hajj et al., 2009). This mismatch tolerance is

useful – especially when sloppy molecular beacon probes are being used to identify

multiple species of bacteria – amplicons from numerous species (El-Hajj et al., 2009).

Finicky molecular beacons, on the other hand, are designed to detect only single-

nucleotide polymorphisms (Tyagi & Kramer, 1996). When detecting single-nucleotide

polymorphisms, it is imperative that finicky probes be used in order to avoid false

positive results.

Figure 1.5: Operation of finicky vs. sloppy molecular beacon probes comparing binding ability of the two types. Modified from Tyagi & Kramer et al., 1996.