Sensitive Detection of BRAF Hotspot Mutation V600E using

E-ice-COLD-PCR Combined with Pyrosequencing

Fei Yu

, Kunxian Shu

and Dan Pu

Chongqing Key Laboratory of Big Data for Bio Intelligence, Chongqing University of Posts and Telecommunications,

Chongqing, China

Keywords: E-ice-COLD-PCR, Pyrosequencing, BRAF, V600E.

Abstract: It is important to sensitively detect BRAF V600E mutation, since it is an important biological marker in several

types of cancers, such as melanomas and colorectal cancers. Here, we have combined enhanced improved and

complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-

COLD-PCR) with pyrosequencing for the detection of BRAF V600E mutation. A serial of mutation-

containing dilutions was determined, and the detect limit of E-ice-COLD-PCR/pyrosequencing and

conventional PCR/pyrosequencing assays were 0.1 and 5%, respectively. When BRAF V600E mutation in 10

melanoma patients were further determined, all samples with the V600E mutation detected by conventional

PCR/pyrosequencing were also found to be detectable with E-ice-COLD-PCR/pyrosequencing. However, one

sample was detected by using E-ice-COLD-PCR/pyrosequencing but not by conventional

PCR/pyrosequencing. In summary, E-ice-COLD-PCR/pyrosequencing is sensitive, reliable and cost-effective

for detecting BRAF V600E mutation in clinical samples.

1 INTRODUCTION

Mutations in BRAF have been found in a wide range

of human cancers, including melanomas (59%),

colorectal cancers (10%), thyroid cancers (30–70%),

and early ovary cancers (30%) (Besaratinia 2008). Of

those mutations, BRAF V600E mutation which

replaces the valine by glutamate at codon 600 is the

most frequent mutation, accounting for ~92% of all

BRAF cancer mutations (Besaratinia 2008). Thus, it

is clinically important to detect BRAF V600E

mutation. Sanger sequencing is the gold standard for

molecular diagnosis, and it is accurate and reliable

(How-Kit 2013). However, it has several limitations

in terms of costs effectiveness and sensitivity. For

example, its limit of detection is only 10–30% of

mutated alleles in a background of wild-type alleles

(How-Kit 2013). Mostly, the DNA abundance or

concentration of specimen samples obtained from

tumor tissues and liquid biopsies, such as cell-free

DNA (cfDNA), may be lower than 1%. In this case,

the low sensitivity of Sanger sequencing will not be

https://orcid.org/0000-0003-3069-0156

https://orcid.org/0000-0003-4487-2727

https://orcid.org/0000-0002-0156-898X

sufficient to detect the aforementioned low-

abundance BRAF V600E mutation.

Pyrosequencing is a real-time DNA sequencing

technique which measures the sensitivities of emitted

light during DNA synthesis. It employs a set of

enzymatic reactions to generate inorganic PPi and to

convert it to visible light during the polymerization.

This technology adds one nucleotide into the reaction

at a time during DNA synthesis. Due to the previously

known on the type of bases added, the sequence of the

determined template can be interrogated sequentially

(Tan 2008). Pyrosequencing offers a specific,

sensitive, rapid and cost-effective alternative to

dideoxy sequencing for the detection of BRAF V600E

mutation (Tan 2008). Although pyrosequencing has a

low detection limit of 5%, it still does not enable to

detect the low percentage of mutated DNA.

To further decrease the limit of mutation

detection, methods which are capable of enriching the

unknown mutations in samples have been developed.

For example, Co-amplification at lower denaturation

temperature PCR (COLD-PCR) has been established

Yu, F., Shu, K. and Pu, D.

Sensitive Detection of BRAF Hotspot Mutation V600E using E-ice- COLD-PCR Combined with Pyrosequencing.

DOI: 10.5220/0011230300003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 535-539

ISBN: 978-989-758-595-1

535

to highly enrich low-abundance mutations from

clinical samples, and the detection efficiency is

independent of the mutation type and position (Li

2008). However, COLD-PCR is not able to

effectively enrich and identify all forms of unknown

mutations (Li 2008, Milbury 2011). Another

drawback of COLD-PCR is that it needs to very

precisely control a critical temperature (T

) as a slight

variation of 0.2°C may completely fail the mutation

enrichment (Milbury 2011). Therefore, by the

inclusive of a wild-type (WT) specific blocker probe,

an improved and complete enrichment COLD-PCR

(ice-COLD-PCR), in which a WT specific blocker

probe was included, has been proposed to eliminate

shortcoming of COLD-PCR that cannot enrich all

mutation types (Milbury 2011). Further, by the

incorporation of locked–nucleic acid (LNA)

nucleotides in the WT blocker probe, a relatively

novel technology, Enhanced-ice-COLD-PCR has

also been developed. It maximizes the T

differences

between homo- and heteroduplexes for a single base

mis-match and thus overcomes the above-mentioned

issue that acquires a critical T

in the COLD-PCR

assays (How-Kit 2013). This technique provides a

sensitive, reproducible and flexible technique for

mutation enrichment.

In the present study, we demonstrate that E-ice-

COLD-PCR is used to enrich low abundance BRAF

V600E mutation and pyrosequencing is then applied

as the downstream readout technology. The

efficiency of E-ice-COLD-PCR/pyrosequencing is

evaluated in detecting BRAF hotspot mutation

V600E.

2 MATERIALS AND METHODS

2.1 Samples and DNA Extraction

Tumor specimens were obtained from 10 melanoma

patients in this study. Genomic DNA from formalin-

fixed paraffin-embedded (FFPE) tissue was extracted

using the QIAamp DNA FFPE Tissue Kit (Qiagen)

following the manufacturer’s instructions. After

extraction and purification, the DNA was quantified

using a Qubit fluorometer (Thermo Fisher, Waltham,

MA).

2.2 Plasmid Standards

Plasmid DNA templates harbored BRAF V600E

mutation in exon 15 of BRAF gene were generated. A

plasmid containing wild-type BRAF exon 15 was also

constructed. The mutation type of plasmids was

verified by using a PyroMark Q96 MD

pyrosequencing instrument (Qiagen, Courtaboeuf,

France). All the plasmids of the defined genotypes

were used to generate amplicons for positive and

negative controls.

2.3 Conventional PCR

Twenty-five nanogram of genomic DNA was used as

template in a 25-μL PCR mix including 1× HotStar

Taq DNA polymerase buffer, 1.6 mM of additional

MgCl

, 200 μM of dNTPs, 200 nM of the forward and

reverse primers, and 2 U of HotStar Taq DNA

polymerase. PCR cycling conditions contained an

initial 95◦C denaturation for 3 min, followed by 40

cycles of 95◦C for 30 s, 56◦C for 30 s, 72◦C for 10 s,

and 72°C for 5 min. Amplicons were verified by gel

electrophoresis on a 2% agarose gel prior to

pyrosequencing.

2.4 E-ice-COLD-PCR

E-ice-COLD-PCR reaction volume was 50 µL and

the reaction mixtures contained 1× of Phusion DNA

polymerase buffer, 25 ng of genomic DNA, 200 μM

of dNTPs, 200 nM of each primer, 1.6 mM of

additional MgCl

, 40 nM of the blocker probe, and 5

U of Phusion

DNA polymerase (New England

Biolabs (NEB)). E-ice-COLD-PCR protocol

contained an initial 95◦C denaturation for 10 min,

followed by 6 cycles of 95◦C for 30 s, 60◦C for 20 s,

72◦C for 10 s, and then 44 cycles of 95◦C for 20 s,

70◦C for 30 s, 60◦C for 20 s, 72◦C for 10 s, and a final

extension at 72°C for 5 min.

2.5 Pyrosequencing

Twenty µL of conventional PCR or E-ice-COLD-

PCR products were purified and the purified products

were applied to generate single-stranded for

sequencing reaction by use of a PyroMark Q96

Vacuum Workstation (Qiagen, Courtaboeuf, France).

Thereafter, the sequencing primer was annealed to the

single-stranded target sequence after incubation at

80°C for 2 min. The order of nucleotide dispensation

was C/A/G/A/T/G/A/T/C. After the run, the

abundance of each genotype was determined by the

AQ model of Pyromark Q96 MA software and shown

upon the pyrogram.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

536

3 RESULTS

3.1 The Determination of T

It is important to determinate the optimal critical T

for E-ice-COLD-PCR reactions, at which mutant

sequences were robustly amplified as well as

Figure 1: Determination of suitable critical denaturation

temperatures (Tc) using a set of E-ice-COLD-PCR assays

with gradient Tc (from 72◦C to 82◦C) on 5% mutation

fraction in mixed plasmids and 40 nM of blockers.

at 5% mutant abundance with 40 nM of blocker

probes. The PCR products were analyzed by

pyrosequencing. As shown in Figure 1, with the

increase of the Tc, the enrichment efficiency of

mutant sequences increased. When Tc was 76°C,

mutant sequences could be effectively enriched.

However, when the Tc is higher than 78.3°C, the

enrichment efficiency of mutant sequences gradually

decreased. Thus, 76°C was chosen as the appropriate

for the E-ice-COLD-PCR in this study.

3.2 Comparison of Standard

PCR/Pyrosequencing and

E-ice-COLD-PCR/Pyrosequencing

for V600E Mutation Detection

To compare conventional PCR/pyrosequencing and

E-ice-COLD-PCR/pyrosequencing in detecting low-

level BRAF V600E mutation, plasmid DNA with

BRAF V600E mutation was diluted serially into wild-

type DNA to generate the following fractions of

mutations: 40, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01 and

0%. pyrosequencing (Figure 2). As shown, the

mutation abundances obtained by both conventional

PCR/pyrosequencing and E-ice-COLD-

PCR/pyrosequencing decreased, as the ratios of the

mutant to wild-type plasmids decreased. Since the

reported detect limit of pyrosequencing was

approximately 5% (Mauger 2017), mutation

abundance lower than 5% was undetectable. Thus, E-

ice-COLD-PCR/pyrosequencing was capable of

detecting BRAF V600E mutation at a much lower

ratio of mutant to wild-type alleles (0.1%) than

conventional PCR/pyrosequencing (5%). That was to

say, the detection limit of pyrosequencing was as low

as 0.1% when combined with upstream enrichment E-

ice-COLD-PCR.

Figure 2: Comparison of conventional PCR/pyrosequencing

and E-ice-COLD-PCR/pyrosequencing for BRAF V600E

mutation detection.

3.3 E-ice-COLD-PCR/Pyrosequencing

for BRAF V600E Detection in

Clinical Samples

To further explore the potential clinical applications of

E-ice-COLD-PCR/pyrosequencing for BRAF V600E

detection in clinical samples, FFPE tissues from 10

melanoma patients were enriched by E-ice- COLD-

PCR followed by pyrosequencing. The same samples

were also analyzed by using conventional

PCR/pyrosequencing for comparison. As shown in

Table 1, the V600E mutations in all the five samples

that were positive by conventional

PCR/pyrosequencing were successfully detected by

using E-ice-COLD-PCR/pyrosequencing.

Furthermore, among the 5 undetectable samples, one

of them with no V600E mutation detected by

conventional PCR/pyrosequencing was successfully

detected by E-ice-COLD-PCR/pyrosequencing. That

was, E-ice-COLD-PCR/pyrosequencing increased the

mutations detected in clinical melanoma specimens by

10% (1/10). Therefore, E-ice-COLD-

PCR/pyrosequencing is more sensitive in detecting the

V600E mutation when compare with conventional

PCR/pyrosequencing, which might assist in cancer

treatment and monitoring and in prenatal diagnosis.

Sensitive Detection of BRAF Hotspot Mutation V600E using E-ice- COLD-PCR Combined with Pyrosequencing

537

Table 1: Comparison of conventional PCR/pyrosequencing and E-ice-COLD-PCR/pyrosequencing assays for BRAF V600E

mutation detection in clinical samples.

No. Mutation Conventional PCR/pyrosequencing E-ice-COLD-PCR/pyrosequencing

S1 c.1799T > A WT

S2 c.1799T > A WT WT

S3 c.1799T > A WT

S4 c.1799T > A MT

S5 c.1799T > A MT MT

S6 c.1799T > A MT MT

S7 c.1799T > A WT WT

S8 c.1799T > A MT MT

S9 c.1799T > A WT WT

S10 c.1799T > A MT MT

The grey highlighted sample was with no V600E mutation detected by conventional PCR/pyrosequencing but successfully

detected by E-ice-COLD-PCR/pyrosequencing. a: Wild-type (WT). b: Mutant-type (MT).

4 DISCUSSIONS

With the introduction of precision medicine, the

ability to detect low-abundance mutation or DNA has

become more and more important in several clinical

areas including diagnosis, treatment and prognosis of

cancers, non-invasive prenatal diagnosis, forensic

identification and so on. Here, we combined E-ice-

COLD-PCR with pyrosequencing to detect BRAF

V600E mutation in serial dilutions of mutant DNA

and in clinical samples. The results demonstrated that

E-ice-COLD-PCR/pyrosequencing showed higher

levels of enrichment and sensitivity over

conventional PCR/pyrosequencing. The detect limit

of 0.1% in this work was consistent with the reported

work for KRAS mutations detection (How-Kit 2013).

However, it was slightly lower than that of the

previously published work in which an E-ice-COLD-

PCR/pyrosequencing assay has been used for

detecting BRAF mutations with a detection limit of

0.01% (How-Kit 2014). The main reason might be

that the different concentrations of blocker probes for

different types of samples or different amounts of

DNA input might show different detection quality

and quantity (How-Kit 2014).

The widely used downstream read-out

technologies includes HRM analysis, Sanger

sequencing, and NGS. These methods are either not

sensitive enough or too expensive when used in

detecting low-abundance BRAF V600E mutation.

Pyrosequencing is relatively sensitive (with a

detection limit of 5%) and cost-effective for mutation

detection. It enables to detect 0.1–0.01% of mutant

DNA, when combined with E-ice-COLD-PCR to

analyze mutations (How-Kit 2013, How-Kit 2014). In

this study, the samples were analyzed in duplicates. A

5% mutation threshold for pyrosequencing was

considered a simple as mutant type corresponding to

the limit of detection of pyrosequencing. The samples

were considered as mutated if their abundances

showed a mutation level higher than 5%. In addition,

when FFPE tissues were analyzed by E-ice-COLD-

PCR/pyrosequencing, one sample with no mutation

detected by conventional PCR/pyrosequencing was

successfully detected by using E-ice-COLD-

PCR/pyrosequencing. The results clearly

demonstrated the power of E-ice-COLD-

PCR/pyrosequencing in BRAF V600E mutation

detection and identification in clinical samples.

5 CONCLUSIONS

All in all, we have combined E-ice-COLD-PCR with

pyrosequencing to detect BRAF hotspot mutation

V600E, and demonstrated that the use of E-ice-

COLD-PCR/pyrosequencing increased the sensitivity

for detecting BRAF V600E mutation with detection

limit of 0.1%. When FFPE tissue specimens were

detected, one sample was detected by using E-ice-

COLD-PCR/pyrosequencing but not by conventional

PCR/pyrosequencing. Thus, E-ice-COLD-

PCR/pyrosequencing is high sensitivity, reproducible

and flexible for identifying low-abundance BRAF

V600E mutation in clinical specimens.

ACKNOWLEDGEMENTS

The work was funded by the National Science

Foundation of China [61801071], and the Basic

Research and Frontier Exploration Project of

Chongqing Science and Technology Commission

[CSTC2018jcyjAX0246].

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

538

REFERENCES

Besaratinia, A., Pfeifer, G.P. (2008) Sunlight ultraviolet

irradiation and BRAF V600 mutagenesis in human

melanoma. Hum. Mutat. 29, 983–991.

How-Kit, A., Mazaleyrat, N., Daunay, A., Nielsen, H.M.,

Terris, B., Tost, J. (2013) Sensitive detection of KRAS

mutations using enhanced-ice-COLD-PCR mutation

enrichment and direct sequence identification. Hum.

Mutat. 34,1568–1580.

How-Kit, A., Lebbe, C., Bousard, A., Daunay, A.,

Mazaleyrat, N., Daviaud, C., Mourah, S., Tost, J.

(2014) Ultrasensitive detection and identification of

BRAF V600 mutations in fresh frozen, FFPE, and

plasma samples of melanoma patients by E-ice-COLD-

PCR. Anal. Bioanal. Chem., 406: 5513–5520.

Li, J., Wang, L., Mamon, H., Kulke, M.H., Berbeco, R.,

Makrigiorgos, G.M. (2008) Replacing PCR with

COLD-PCR enriches variant DNA sequences and

redefines the sensitivity of genetic testing. Nat. Med.

14, 579–584.

Milbury, C.A., Li, J., Makrigiorgos, G.M. (2011) Ice-

COLD-PCR enables rapid amplification and robust

enrichment for low-abundance unknown DNA

mutations. Nucleic Acids Res. 39, e2.

Mauger, F., How-Kit, A., Tost, J. (2017) COLD-PCR

Technologies in the Area of Personalized Medicine:

Methodology and Applications. Mol Diagn Ther. 21(3),

269-283.

Tan, Y.H., Liu, Y., Eu, K.W., Ang, P.W., Li, W.Q., Salto-

Tellez, M., Iacopetta, B., Soong, R. (2008) Detection of

BRAF V600E mutation by pyrosequencing. Pathology.

40, 295–298.

Sensitive Detection of BRAF Hotspot Mutation V600E using E-ice- COLD-PCR Combined with Pyrosequencing

539