Detection of p53 gene single nucleotide polymorphism

in breast cancer cell line (MCF-7) using Polymerase

Chain Reaction and Restriction Fragment Length

Polymorphism

Farhath JabienUniversity Bradfordemail contact: J.Farhath@bradford.ac.uk

Abstract: Background: to detect any polymorphism in p53 gene. Methods: Trypsinization,

Quantification, Isolation of DNA, gel electrophoresis, PCR, RFLP were performed.

Results: There were visible bands on agarose gel (3%) after RFLP. Conclusion: There is a

polymorphism in p53 gene sequence where Arg became Pro due to missense mutation

between Guanine and Cytosine. This was confirmed by Restriction Enzyme BstUI

fragmentation.

Introduction:

p53 or known as TP53 gene is a tumor suppressor gene, encoding for tumor suppressor p53

protein. It plays critical role in cell cycle control, initiation of apoptosis and DNA repair. A

single nucleotide polymorphism can lead to severe or fatal complications like cancer. One of

the most common cancers associated with p53 mutation is breast cancer. (Hu,Y.,et al,2005)

A single nucleotide polymorphism (SNP) can be found be coding region, non-coding or

intergenic (coding between genes) regions of genes. Any of the four nitrogenous bases (A,

T,G,C) could be changed to another. As such, if the mutation does not cause any change in

polypeptide produced (amino acid), it is known as silent mutation or synonymous

polymorphism. However, if there is a change in polypeptide produced, it becomes

replacement mutation or non-synonymous polymorphism.  A replacement polymorphism

change may be either missense, which results in a different amino acid, or nonsense, which

results in a premature stop codon. Most diseases like arise from replacement mutation. Thus it

is important to study polymorphism.

In this experiment, MCF-7 cells lines were used. It is a cancer cell line obtained from a 69-

years old woman. MCF stands for Michigan Cancer Foundation-7, an institute in Detroit,

USA. It is now known as Barbara Ann Karmanos Cancer Institute( Glodek,C.,1990)

Materials and Methods:

Cell culture and reagents

MCF-7 cell line (catalogue no. HTB-22) was purchased from American Type Culture Collection(ATCC) and was provided by the School of Life Science department in MDIS. Promega DNA kit (catalogue no. A1120) consisting of RNAse,Nuclei Lysis Solution,Protein Solution,DNA Rehydration Solution was used for Isolation of DNA. Fermantas PCR-Master Mix( catalogue no. K101) consisting of Taq polymerase (pH 8.5), Nucleotides(dATP,dGTP,dTTP,dCTP) and MgCl2 was used for PCR. 1st Base primers (catalogue no. 123755)consisting of 72F,72R,72_1414R,217F,217R,267F,267R,278F,278R,290F,290R1,290R2, 360F,360R was used for PCR as well. BstUI from Promega was used for Restriction Enzyme.

Trypsinization:

The sole purpose was to dislodge the adhered MCF-7 cells from the T25 flask. Prior to this, culture media and trpsin was incubated. Media was aspirated and PBS was added to remove excessive bovin serum. After removal of PBS, trypsin was added to dislodge the cells and media was added to neutralise and disperse the cells. The media was centrifuged to pellet cells to be used later in other processes.

Isolation of DNA:

This process required first addition of PBS to wash the cells in tube and removed. The cell pellet was resuspended in Lysis solution to allow contents to disrupt the membrane and to denature the proteins.RNAse was added to remove RNA and Protein was precipitated out using Protein Precipitation Solution. After drying the tube, DNA Rehydration Solution was added and incubated for further use.

Quantification of DNA:

In order to know if the DNA isolated is pure from impurities like protein and RNA and cell lysates, spectrophotometry was performed under 2 wavelengths of 260nm and 280nm. Firstly, the DNA sample was diluted to 100-fold and using a quartz cuvette, absorbance was measured. The purity ratio and the concentration of DNA in the sample was calculated before assuming it is possible to proceed on further.

Polymerase Chain Reaction and Gel Electrophoresis:

In order to amplify the specific codons to which the chosen primers (mentioned above) bind to, PCR was performed. Using the Master mix from Fermentas and primers, 2 PCR machines were set up at different temperatures(55oC and 60oC) as the annealing temperatures by primers were not the same. After the end of reaction, the PCR products were analysed in Gel

electrophoresis (1% agarose). The bands obtained were studied under Polaroid Documentation Camera and were compared with DNA ladder to measure sizes.

Restriction Enzyme:

Restriction Fragment Length Polymorphism (RFLP) was performed on codon 72 to analyse if there is any polymorphism. This was done with the aid Gel electrophoresis. The enzyme used was BstUI and was first incubated in water bath at 37oC for 3 hours. Due to unsatisfactory results, experiment was repeated again. This time incubated at 60oC for 4 hours. Results were studied under Polaroid Documentation Camera.

Results & Discussion:

1) Quantification of DNA using spectrophotometer and nano-drop

Absorbance at 260nm wavelength (spectrophotometer): 0.073

Absorbance at 280nm wavelength (spectrophotometer): 0.047

Pure of DNA solution(ratio)OD260nm/OD280nm ~1.8-2.8

Purity of sample DNA (spectrophotometer) = 0.073/0.047 = 1.6

Absorbance at 260nm wavelength (nano-drop): 0.437

Absorbance at 280nm wavelength (nano-drop): 0.241

Purity of sample DNA (nano-drop) = 1.811

Concentration of unknown sample DNA (spectrophotometer) = 50ug/ml X OD260 *X dilution factor

= 50 X 0.073 X 100 = 365ug/ml = 0.365ug/ul *OD260nm (~1.0 reading is equivalent to 50ug/ml)

Concentration of unknown sample DNA(nano-drop) = 436.724ng/ul

DNA quantification can be done with spectrophotometric measurement of UV absorbtion at

260nm and 280nm. At 260nm, DNA absorbs light most strongly. However,since RNA and the

aromatic rings of proteins have strong absorbance at 260nm, the value could be

overestimated. Wavelength 280nm is used concurrently, as the aromatic rings of proteins

absorb at this wavelength. Thus, that OD280nm has to be as low as possible, to conclude that

the DNA isolated is pure for other procedures(Stoscheck,C.M.,1990)

The absorbance values denote the amount the ultra-violet irradiation the nitrogenous bases in

DNA absorbs. The purity ratio calculated above checks that the DNA is pure and that it

contains insignificant amount of contaminants such as proteins, celluar lysates, phenols

carbohydrates. The results obtained from this experiment show little deviation from the

standards. Thus, it can be safely concluded that the DNA to be used consequently was

obtained with no contamination to affect other results that follows. (Vinod,K.K.,2004)

The spectrophotometer and nano-drop spectrophotometer has different precisions and certain

drawbacks. Thus, the results might vary.

The spectrophotometer required certain dilution to allow sufficient liquid in the cuvette

before the machine could pass the wavelength to deliver the absorbance. It could be that the

distilled water used for dilution might have impurity or the cuvette sides where the radiation

passes through might not have been wiped properly to prevent any disruption to the path of

rays. Also, there could have air bubbles present in cuvette that could have dirupted the rays

from passing through the cuvette. On the other hand, the nano-drop machine measures nano

amount of DNA without any dilution. Thus it can detect more precisely with little room for

discrepancies.

2) Polymerase chain reaction(PCR) and gel electrophoresis:

In order for the primers to function, they have to work under their optimum anneling

temperatures. The formula being: T annealing =( Tm forward + Tm reverse /2)-5 was used to calculate

the various annealing temperatures of different primers as seen in Table 2. Table shows the

individual melting temperatures of the primers.

Table 1: Melting temperatures of individual primers

Primer set Annealing temperature(oC):

(Ta={[TmF + TmR]/2}-5

47F+47R 56.6

72F+72R 63.3

72F+72_1414R 62.6

217F+217R 65.15=65.2

267F+267R 56.15=56.2

278F+278R 47.8

290F+290R1 47.8

290F+290F2 45.0

360F+360R 61.65=61.7

Table 2: Annealing temperatures of primers

An example of calculation would be as following:Tannealing =( Tm forward + Tm reverse /2)-5

= (64.5 +58.7)/2 -5 =56.6oC

Primer Set Primer Sequence Expected Size of Product (bp)

Section of p53 gene

47F and 47R tga gga cct ggt cct ctg ac

gag gaa tcccaa agt tcc aaa

412 11832 – 12243

72F and 72R tcc ccc ttg ccg tcc caa gc

cgg cca ggc att gaa gtc tca tgg

328 11874 – 12201

72F and 72_1414R tcc ccc ttg ccg tcc caa gc

agg cgg ctc ata ggg c

1411 11874 - 13284

217F and 217R cgc gtc cgc gcc atg gcc

cgc cca gcc aag cag ggg

496 13000 – 13495

267F 267R cac cct gca cac tgg cct

gct aca acc agg agc cat

778 13982 – 14759

278F 278R ctt ctc ctc cac cta cct

cac ttg ata aga ggt ccc

479 14163 – 14641

290F 290 R1 ctt ctc ctc cac cta cct

cac ttg ata aga ggt ccc

479 14163 - 14641

290F 290R2 ctt ctc ctc cac cta cct

agt caa gaa gaa aac ggc

524 14163 – 14686

360F 360R act tga acc cca gag gcg

cac tcg cct tgg cct ccc

829 17172 - 18000

Table 3: Sizes of bands of PCR-products. Source: Stothard,P., (2000)

Figure 1A: Polaroid Documentation of gel electrophoresis of PCR-products. note: codon 72 and codon 360 are missing.

Discussion:

PCR was developed by scientist Kary Mullis in 1983. The difference in charge and size of the

molecule leads to separation of its components. The DNA molecule being negative charge

runs towards the positive anode from negative cathode end. The various band size( table 3)

are due to difference in fragment sizes being trapped in gel pores. In this experiment, PCR

was used to amplify the specifc regions/codons seen from Figure 1A. In this way, any

polymorphism of codons can be detected if the specific sizes of bands are not obtained in the

gel after electrophoresis. For instance, if polymorphism occurred in codon 290* , the size of

band would not be 524bp. In this experiment, most of the bands were obtained at right sizes

with exception of 2 codons. This proves that PCR was successful for most codon

amplification.

Codon290*

Codon290

Codon278

Codon267

Codon217Codon72

Codon47

DNA ladder 1kB DNA ladder 1kB+

412bp 328bp 496bp 778bp 479bp 479bp 524bp

5000400030002000150010007005004003002007520000100007000

100008000600050004000350030002500200015001000750500250

In the gel, there is ethidium bromide present, that would intercalate with the double stranded

DNA, between base pairs, to give a visible band. High molecular weight DNA will appear

well-resolved band alongside with the DNA ladder which consists of uncut lamda DNA. The

gel was prepared only when PCR products were almost ready to be analysed in order to

prevent dryness and loss of pore sizes.Moreover, leaving the gel in the buffer would allow

salt formation. This is unwanted.

In figure 1A, there are 2 missing codons that have failed to amplify. There could be several

possibilities:

a) Primers must not anneal to other primers including themseleves or reverse

primers.There could have been dimerisation between primers of 72F and 72_1414R

for codon 72 and 360F and 360R for codon 360. Such dimerisation leads to

contamination in mixture.

b) The DNA molecule could be too small that it dissolved in the gel or it could be too big

that it would have flowed out and mixed with the buffer.

c) For gel electrophoresis, TAE buffer was used, which has lower buffering capacity and

could be exhasued during electrophoresis. This would lead to the anode becoming

more alkaline and cathode being acidic. The resolution of the picture in figure1A

could have been sharper if TBE buffer was used. Thus, if the band was formed, it

could have been noticed.

3) Restriction enzyme and gel electrophoresis:

Figure 1B: Restriction enzyme(BstUI) gel electrophoresis on codon 72(3% agarose)

During the 1st trial, there were no visible bands for any of the groups. This was because, due

to large dilution, the amount of DNA concentration became insignificant to be detected and

be cut by the restriction enzyme.The following procedures were carried out:

Firstly, PCR reaction 2 was quantified after 1000fold dilution and the DNA quantity was

calculated to be 0.45ug/ul. As stated in Fermentas, the DNA concentration was to be between

0.1-0.5ug of DNA in 10ul. Thus using C1V1 = C2V2, the volume of DNA to be used was

1.2ul of PCR product along with 8.8ul of nuclease free water. This was incubated for 3 hours

in water bath at 37oC. However, when gel electrophoresis (2% agarose) was performed, no

visible bands were seen for any group.

In the 2nd experiment, the DNA was not diluted. 15ul of undiluted PCR-2 reaction mix was

used with doubled amount of enzyme from 1ul to 2ul. This time, it was incubated at 60oC for

4 hours. The temperature was increased significantly as the enzyme is heat resistant and it is

obtained from Geobacilus stearothermophilus.

120bp

200bp

328bp

DNA Ladder 100bp

DNA ladder 1kB

Figure 1B, shows the results of 2nd trial. The restriction enzyme BstUI has cut the codon 72

into 3 fragments as seen. There is evidence of RFLP in only 1 lane. Lane 9 sample belonged

to group 4 (Farhath and team). Other lanes were shared by other groups due to shortage of

time. There seems to be no visible bands on other lanes. This could be due to contamination

in PCR 2 product,

In theory, it is known that p53 Proline (Pro) found in allele has unique site for BstUI site to

cut unlike Arginine (Arg) (Chua,W.H. et al.,2010) The results in 1B shows that there are 3

digested fragments, 328bp,200bp and 120bp. This means there could be possible mutation in

base to change Arg to Pro and cause the enzyme to act. The bands obtained were closely

resembling to the experiment done by Indian scientists in South India by Vijayaraman K.P.,et

al, 2012. The fragment sizes they obtained were 174 bp,135 bp and 309 bp.

Conclusion:

It was found that codon 72 is the codon that is most susceptible to polymorphism, where Arg

becomes Pro. The Arg amino acid encodes for the following codes, cga, cgc, cgg and cgu

whereas the Pro amino acid encodes for the following codes, cca, ccc, ccg and ccu.

(Tong,D.,2000) Since the BstUI enzyme acted on codon 72, it can be said that there is a

missense mutation between Guanine and Cytosine nitrogenous bases.

Though codon 72 has been the most susceptible codon for polymorphism, there are other

codons we should be concerned about and scientists have done several researches about.

For instance, for Breast cancer, the Arginine in codon 72 has no association with the cancer ,

but it is seen to be more prone to degradation by Human papilloma virus that causes Cervical

Cancer. Polymorphism on codon 279, where Serine (Ser) becomes Arginine, shows that there

is missense mutation between Cytosine from Arg becomes Uracil in Ser and Guanine from

Arg becomes Cytosine in Ser( Vijayaraman, K.P.et al,2012)

Moreover, it was found that codon 47 undergoes polymorphism, however,rarely, causing Pro

to become Ser. This is more common in the African Sub-continent. This mutation acts as

hindrance for p53 to cause apoptosis. (Li,X.et al,2005)

Besides RFLP, there are several methods used to detect p53 polymorphism. One such

example are :

a) Gene sequence mutation analysis of p53 gene on lung cancer by direct

dideoxynucleotide sequencing or via Gene ChIP assay. 1.8 kb fragment was used

from p53 gene (exon 5-9). It was amplified by primary tumor DNA by

PCR,electrophoresized in 6% SDS PAGE and exposed to film. Exon 2-11 of p53 gene

was sequenced by gene ChIP assay , detects an alternate form of gene muatation. This

showed that all p53 mutation lead to amino acid substitution, or by insertion/ deletion.

This was true for most except codon 47 and 72.

Mutation specific oligonucleotide hybridisation was used to further identify the mutation and

tumor. After RFLP, the amplified p53 was cloned onto a vector and amplified in Ecoli ( fast

replication). They were transferred to nylon membrance and hybridised using 32P-end labelled

oligonucloetide probe specific to tumor. Hybridisation would occur only if there is a missense

mutation.