Identification of single nucleotide polymorphism in the p53 gene of

breast cancer cell lines

Nang Hom Khan, Nan Chan Myae, Thandar Soe, May Tun Myat Oo (or) Aye Nyein Su.
Bradford University(UK), Management and Development Institute of Singapore(MDIS).
UOB: 12038200. Batch number: BBSD11318A
Email: N.H.Khan1@student.bradford.ac.uk

Abstract
Single nucleotide polymorphism(SNP) is associated with single base changes in
DNA sequence and it accounts for one percent within the population. Approximately of
ten millions SNP exists in the genome of human because on average SNP is commonly
presented in every three hundred of nucleotide. If SNP occurred in a particular important
gene, it can lead to the alteration of the function of that gene. The p53 protein, translated
from p53 gene, function to protect the cell against all cellular stresses. SNP is commonly
occurred at codon 72 of p53 gene and alter the protective activity of this gene and is
associated with various types of cancer in human. In this experiment, MCF-7 and MDA-
MB-231 breast cancer cell lines were used to access the presented of SNP at codon 72
and mutation at codon 280 of p53 gene. First harvesting of both of these cells lines were
done using cell scrappers followed by extraction of genomic DNA with promega wizard
kit. The present of extracted DNA was confirmed with agarose gel electrophoresis and
the targeted sequence was amplified with PCR for both cell lines. Two set of primer cell
lines were used to amplified MCF-7 cell lines which resulted in two targeted sequence
328bp and 1412bp. Restriction enzyme digestion was done for both MCF-7 targeted
sequence and SNP at codon 72 was figured out by comparing the restriction fragments of
targeted sequence with wild type p53 gene restriction fragments. Mutation at codon
280(AGA to AAA) was confirmed by sequencing.
Key words: p53, MCF-7, MDA-MB-231, SNP, breast cancer, codon 72 and 280.

Introduction
Breast cancer is one of the most common malignancy encountered in women around
the world and sporadic breast cancer accounts for more than 90% of all cases. Although,
several risk factors such as genetic, environmental factors, intake of diet that contains
high fat, family history, vitamin D deficiency and oral contraceptive consumption have
suggested to be involved in developing of breast cancer. The p53, a tumor suppressor
gene (OMIM entry):*191170, located on chromosome 17p13.1 and consists of 11 exons.
This gene product have been known as the guardian of the genome, function to regulate
the cell cycle, deoxyribonucleic acid(DNA) repair and inducing apoptosis in response to
cellular stresses. p53 gene levels and activity are very low in normal healthy cell. Several
mutations and single nucleotide polymorphism(SNP) exist in p53 gene. Mutation in p53
tumor suppressor gene is associated with 50% of human cancer including breast cancer
and elevates its risk. Mutations are mainly found in DNA binding domain of p53 gene
and lead to a partial or complete loss of transactivation functions. SNP is associated with
changes in a single DNA base pair(bp). This genetic variations(1%) are commonly
occurred within a population. The p53 gene SNP at codon 72 in exon 4 is frequently
studied one. SNP leads to either arginine(Arg) CGC or proline(Pro) CCC alleles at codon
72, alter the p53 gene function and is involved in an increased risk of cancer in
individual. These two alleles result in three different genotypes (Arg/Arg, Arg/Pro,
Pro/Pro). In our mini research project, we aim to investigate whether MCF-7 and MDA-
MB-231breast cancer cell lines were presented with polymorphism at codon 72 (for
MCF-7) and mutation at codon 280 (for MDA-MB-231) in p53 gene. The p53 SNP at
codon 72 and 280 were investigated by experimental methods such as DNA extraction of
provided cell lines, checked for extracted DNA on agarose gel, performed polymerase
chain reaction(PCR) followed by using agarose gel electrophoresis to identify PCR
product. Restriction enzyme digestion of codon 72 and checked on agarose gel. Mutation
at codon 280 can be determined through DNA sequencing.

Results
Genomic DNA extraction
Provided cell lines were harvested prior to DNA extraction, cell lines MCF-7 and
MDA-MB-231 (which were growth in culture media containing fetal bovine serum and
antibiotics) in T75 flask each were visualized under the inverted phase contrast
microscope to check for (80-85%)confluence of cells. MCF-7 cells looked cobblestone-
like appearance and MDA-MB-231 cells looked spindle shaped , lack of bacteria, fungal
contamination, healthy growing and were approximately 80-85% confluence observed
under the microscope. (Fig.1). The cells from these two cell lines were then dislodged
from adherent at the bottom of the flask mechanically by sterile cell scrapper. Detached
cells were seen as spindle shaped and floating when examined under the microscope in
the suspension.(images have not included).

Figure 1. MDA-MB-231 (left figure) spindle shaped cells MCF-7 (right figure)
cobblestone like shape (80-85%) confluence when visualized under the inverted phase
contrast microscope before cell were harvested.
 Genomic DNA extraction of both cell lines were performed using Promega Wizard
kit protocol. The ideas of this principle involved lysis of the cell membrane with nucleic
acid solution to expose of all the organelles inside the cell including DNA, RNA and
protein. RNA and proteins(unwanted part) were then broken down with RNase and
protein precipitation solution. Isopropanol was used to precipitate DNA and then washed
with alcohol afterward, followed by added the rehydration solution to rehydrate the DNA.
1% agarose gel electrophoresis was then used to check for the presented of genomic DNA
(or) whether genomic DNA was successfully extracted from the cells. After
electrophoresis, the gel was then visualized under the blue light as safe green reagent was
already incorporated with DNA sample before loaded into the well and the result showed
that the genomic DNA was obtained.(data not shown)

Quantification of DNA
Genomic DNA Absorbance Absorbance DNA 260:280
260 280 concentration ratio
ng/µl
MCF-7 2.002 1.032 2001.78 1.94
MDA-MB-231 0.318 0.161 317.93 1.98

Table 1. Quantification of DNA - MCF-7 and MDA-MB-231 cell lines genomic DNA
were quantified by Epoch multivolume spectrophotometer. The absorbance
ratio(260/280) and DNA concentration of MCF-7 were 1.94 and 2001.78ng/µl
respectively whereas MDA-MB-231 were 1.98 and 317.93ng/µl respectively.

Epoch multivolume spectrophotometer and biotek Take3 plate were used to

quantified extracted genomic DNA from both cell lines. The DNA volumes used were 2µl
from each cell line. The purpose of quantification of extracted DNA was to check the
purity and concentration of it. The absorbance of 260nm and 280nm were used because
DNA absorb maximum light at 260nm wavelength, while protein absorb light at the
wavelength of 280nm. After quantification, the results of the absorbance of MCF-7 at
260nm and 280nm were 2.002 and 1.032 respectively, whereas MDA-MB-231 were
0.318 and 0.161 respectively.(Table.1) As the ratio of the absorbance at 260/280 was used
to check the purity of DNA. From the results, DNA of MCF-7 at the wavelength of
260/280 ratio had an absorbance of 1.94 and MDA-MB-231 was 1.98.(Table.1). We
considered a pure DNA was obtained because at A260/280 ratio 1.8 to 2 is generally
accepted as pure for DNA. MCF-7 DNA concentration was 2001.78ng/µl and MDA-MB-
231 was 317.93ng/µl.

Polymerase Chain Reaction

After the presented of the extracted genomic DNA from both cell lines was
confirmed with 1% gel electrophoresis, targeted sequence from both cell lines were
amplified with PCR. The principle behind this technique was to amplify a specific
sequence of DNA using a set of forward and reverse primers, thermal stable DNA

polymerase(Taq polymerase), four different types of deoxynucleotide

triphosphates(dNTPs). Total of three main steps repeating were involved in PCR,
denaturation of double strand DNA at 94°C, annealing of primer at 55°C and extension of
DNA at 72°C. PCR thermal cycler machine was used to amplified a targeted sequence of
both cell lines. SNP at codon 72 was analyzed with one 72 forward(F) primer, and two
reverse(R) primers.
Forward primer Reversed primer
MCF-7(72F+72R) 5’ TCC CCC TTG CCG 5’CGG CCA GGC ATT GAA
TCC CAA GC 3’ GTC TCA TGG 3’
MCF- 5’ TCC CCC TTG CCG 5’ CTC AGG CGG CTC ATA
7(72F+72_1414R) TCC CAA GC 3’ GGG 3’

MDA-MB- 5’CTT CTC CTC CAC 5’ CAC TTG ATA AGA GGT
231(278F+278R) CTA CCT 3’ CCC 3’

Mutation at codon 280 of MDA-MB-231 cell line was amplified with 278 forward primer
and reverse primer. PCR reaction was performed for 35 cycles. The expected fragment of
MCF-7 amplified with 72F and 72R primers were 328 base pair(bp), for 72F and
72_1414R primers were 1412bp. 479bp was an expected fragment for MDA-MB-231,
predicted from serial cloner software (Table 2). From the experiment, a clear band of
PCR product was obtained for MCF-7 in lane 3 (346bp) and MDA-MB-231 in lane 5
(603bp), the deviation of the experimental fragments from the expected one can be due to
electrophoresis conditions used was improper(high voltage and temperature, buffering
capacity of buffer used was insufficient) MCF-7 that was amplified with 72F and
72_1414R primers showed two faint bands in lane 4, the upper band which when added
got 1413bp (Fig.2). In theory, only one band should be resulted in PCR product that
amplified with 72F and 72_1414R primers which was 1412bp, the resulted in two faint
bands can be due to non-specific or incomplete binding of the primer to the template
DNA or during the PCR reagent mixture contamination was introduced by using water
instead of nuclease free water and causing DNA to be degraded. The experimental
fragments size in base pair of lane 3,4 and 5 depicted in(Fig2, Table2) were derived from
blotting a standard curved of migration distance vs log of DNA base pair of known 1kb
and 1kb plus DNA ladder.(Fig.3).

Figure 2. PCR products

were analyzed on 2%
agarose gel
electrophoresis - 1
kilobase (kb) DNA
ladder is shown in lane
1. lane 3 and lane 4
fragments represented
MCF-7 targeted
sequence amplify with
72 forward+ reverse
primers and 72
forward+ 72_1414
reverse primers respectively. MDA-MB-231 targeted sequence was amplified with 278
forward and reverses primer shown in lane 5. DNA ladder of 1 kilobase plus is shown in
lane 7.

Expected fragment of Experimental fragment of

PCR product PCR product
MCF-7 (72F+72_1414R) 1412bp 1413bp
MDA-MB-231(278F+278R) 479bp 603bp
MCF-7(72F+72R) 328bp 346bp

Table 2. PCR fragment in base pair of MCF-7 and MDA-MB-231 cell lines - the
expected fragment of PCR product of MCF-7 and MDA-MB-231 were obtained from
serial cloner software by inputted of p53 gene sequence from nation center for
biotechnology information (NCBI). Experimental fragment of PCR product was obtained
by blotting the standard graph of known 1kb and 1kb plus DNA ladder against distance
migration of it.

Base pair in Log base Distance travelled by Distance travelled by

1kb+1kb plus pairs 1kb+DNA 1kb DNA ladder(mm)
DNA ladder ladder(mm)
1500 3.18 24 69
1000 3 30 51
700 2.85 37 40
500 2.7 42 36
300 2.48 52 51
100 2 72 69

Figure 3.Standard graph to predict unknown DNA base pair for PCR product- Distance
migration of 1kb(red line) and 1kb plus(blue line) DNA ladder in milliliter(mm)
measured from (Fig2) in Y-axis vs log of DNA base pair in X-axis.
 Restriction fragment length polymorphism(RFLP) of codon 72
To verify the presented of SNP at codon 72 of p53 gene, BstU1 restriction enzyme
was used to digest the PCR product. CGCG was the recognition size of the this enzyme
and cut between CG and CG. When 72F+72R primers amplified PCR product was
digested with enzyme in serial cloner software, two expected fragments were obtained,
210bp and 118bp. In our gel results, we also got two bands of DNA after digestion in lane
3 (Fig.4) which were 126bp and 219bp, derived from known DNA ladder(Fig5). The
experimental and expected fragments base pair were quite closed. The deviation of the
experimental fragments form expected one can be due to electrophoresis conditions used
was improper(high voltage and temperature, buffering capacity of buffer used was
insufficient, DNA was denatured). The expected restriction fragments for 72F+72_1414R
primers amplified PCR product were 1007bp, 281bp, 118bp and 6bp. In lane 8 gel results,
all the bands were very faint, only two faint bands were seen 1202bp and
191bp(Fig4).The faint and missing band can be because of not enough PCR product was
loaded for digestion or degradation of DNA occurred. The experimental fragments were
derived from the known DNA ladder standard graph depicted in(Fig.5). As we compared
the expected restriction fragments of 72F+72R primer amplified product with the
experimental one(Table.3) we can say that SNP was presented at codon 72 of p53 gene
and the genotype was arginine homozygous because enzyme will recognized at CG which
was arginine and two fragments was resulted after digestion. While the expected and
experimental fragment of 72F+72_1414R primers were different, the expected fragments
were four and the experimental fragments were three. The experimental fragment resulted
in three bands (Fig4) because SNP occurred within the amplified DNA sequence and lead
to the changes of nucleotide base which the enzyme was not recognized and remained
uncut and leaved larger band on top of the gel. The reason for only two bands was
presented on the gel in lane 8(Fig4) was because the third fragment was very small(79bp)
and electrophoresed off the gel. The genotype of this SNP would be arginine/proline
heterozygous.

Expected restriction Experimental

fragment restriction fragment
MCF-7(72F+72_1414R) 1007bp,281bp,118bp,6bp 1202bp, 191bp,79bp

MCF-7(72F+72R) 210bp, 118bp 219bp, 126bp

Table.3: Expected and experimental restriction fragments of MCF-7 cell line after BstU1
digestion. Expected fragments were generated by serial cloner software while
experimental fragment were derived from blotting the graph of known DNA ladder.

Figure. 4: 3%
agarose gel
electrophoresis used
to run RFLP of
MCF-7 cell line
digested with BstU1
restriction enzyme -
Lane 1( 1kb DNA ladder), lane 3 (two fragments) and lane 8(three fragments) , Lane 10(1
kb plus DNA ladder)
Figure.5:Standard graph to predict unknown DNA base pair of restriction fragment
length - Distance migration of 1kb(red line) and 1kb plus(blue line) DNA ladder in
milliliter(mm) measured from (Fig.4) in Y-axis vs log of DNA base pair in X-axis.

Sequencing of codon 280

PCR product amplified with 278F+278R primers was sent for sequencing to detect
mutation at codon 280 because there was no restriction enzyme that can recognize and
digest this site. From nucleotide blast results, there was a mutation occurred at nucleotide
175 of codon 280 which lead to single base pair changes from G to A(Fig.6A).
Sequencing results also showed that changes of single base pair occurred at nucleotide
175, from AGA to AAA (Fig.6B).
(A)
(B
Figure.6. Comparison of sequencing result of codon 280 with and without mutation -
(A) nBlast results from NCBI showed that a mutation occurred at nucleotide 175 of
codon 280,indicated with arrow, G to A changes. (B) the bottom part of the
chromatography at nucleotide 175 which shown AGA normal codon without mutation.
The top part of the graph, at the same 175 nucleotide as the bottom part which shown
AAA.
Discussion
The objective of this experiment was to investigate whether SNP at codon 72 and
mutation at codon 280 was presented in p53 gene by using MCF-7 and MDA-MB-231
cell lines. MCF-7, human breast cancer cell line was derived from a caucasion women
who was 69 years old and established in 1973 for studying the gene expression in breast
cancer. Inactivation of p53 gene happened when SNP occurred in this gene. Increasing in
the risk of lung, breast, thyroid, bladder and gastric have been associated with p53 gene
polymorphism especially at codon 72. In this experiment, PCR was performed to
amplified the targeted sequence of MCF-7 and MDA-MB-231 cell lines and digestion of
MCF-7 cell line with BstU1 enzyme was carried out to find out SNP at codon 72 and
examined on agarose gel.As mention in introduction section, there were three possible
genotypes which were arginine homozygous, arginine/proline heterozygous and proline
homozygous, individual with specific genotype was susceptible to an increased risk of
cancer. From the experiment, SNP was presented in p53 codon 72 as mention in result
section and it genotype was with arginine homozygous for 72F+72R primers amplified
PCR product and arginine/proline heterozygous was presented in 72F+72_1414 primer
amplified PCR product.(Fig.4). Some study had been done on breast cancer also indicated
that there was SNP at codon 72 of p53 gene and this research paper also indicated that
arginine homozygous was associated with an increased risk of breast cancer while on the
other hand some study showed that proline homozygous had an increased of breast
cancer. Some study indicated that Arginine/proline genotype was associated with a poor
prognosis in breast cancer. Arginine homozygous genotype also elevated the risk of oral
cancer in Taiwan and India according to two research published paper. The controversial
still remains about proline or arginine allele at codon 72 and which allele was more
involved in the risk of cancer and prognosis. Study has been done by two research group
on lung cancer indicated that proline homozygous at codon 72 was linked to an increased
risk of it. Mutation in p53 gene not only lead to the loss of its tumor suppressive activity
but can also obtained an additional new oncogenic activity which is gain of function and
associated with the development of cancer and metastasis. p53 gene mutation at codon
280 was detected in MDA-MB-231 in our experiment by sequencing which showed a
missense mutation by the replacement of AGA to AAA( arginine to lysine)(Fig.6). Some
study has shown that mutant p53 gene which resulted from arginine to lysine substitution
induces Myoxin-X, motor protein and involved in an invasion of breast cancer. While
other study showed that Gaucher disease has been found with arginine to lysine missense
mutation at codon 280.
Conclusion
In our experiment, two breast cancer cell lines had been used to study polymorphism
at codon 72 and 280 of p53 gene. PCR-RFLP and agarose gel electrophoresis methods
were employed in detecting polymorphism at codon 72 and sequencing for codon 280.
PCR-RFLP is the most commonly used method and quite sensitive in identifying SNP.
Polymorphisms were detected at codon 72 with arginine homozygous and
arginine/proline heterozygous genotypes and mutation at codon 280. But only one sample
was used for each cell line in this experiment and the resulted will not be reliable. More
cell lines or larger samples size could be used to produce an accurate results.
Acknowledgement
We would like to thank our supervisor Ms Heather Bennedict Hilminton for
supervising our project and all the laboratory staffs for their assistance.