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The early onset breast and ovarian cancer is often associated with BRCA1. The BRCA1, responsible for the cell growth control is a tumor suppressor, which also functions in the maintenance of the genomic stability. 24 exons and 1863 amino acids constitute the human BRCA1 gene. At the amino terminus, it contains an increasingly conserved RING figure domain with the repetition of the 2 BRCT (BRCA1 carboxyl-terminal) at the carboxyle terminus. The identification of more than 850 clinical mutations in the BRCA1 gene has been established. Many of these genes are held responsible for the increasing risk of breast cancer.
The female population is suffering from breast cancer which is the major reason behind the deaths among the female population. The mutation of the breast cancer susceptibility genes BRCA1/21 is the cause of breast cancer although 5-10% of all the breast cancers are inherited. The identification of BRCA1 was done through genetic linkage analysis and positional cloning in 1994. The location of BRCA1 gene is on position 21 of the long (q) arm of chromosome 171. To be precise the BRCA1 gene is positioned from the base pair 41,196,311 to base pair 41,277,499 on chromosome 172. The frequency at which the germ-line mutation o BRCA1 occurs is 1 in 250 women. These mutations are responsible for the 45% of the familial breast cancers, while 80-90% of the hereditary cases are involved for both the ovarian and breast cancers (breast- ovarian cancer syndrome).3
Convincing evidence has been brought in the lime light about the mutations of BRCA1 responsible for breast and ovarian cancers. Observations obtained initially from the predisposition of the mutations in 5 out of 8 subjects indicated BRCA1 linkage. 4 The evidence was further strengthened by the presence of germ line mutations in 4 out of 44 patients. These patients possessed the putative sporadic breast or ovarian cancer5. Recently a study was conducted on 100 families having a history of multiple cases of breast and ovarian cancer. Among these 31 cases of BRCA1 mutations were revealed in which 22 were the distinct type6. The BRCA1 emerged as a tumor suppressor in the analysis of the BRCA1 associated tumor specimen. The wild type copy of the BRCA1 is dropped by the tumor invariable, while the inherited mutant copy (loss of heterozygosity; LOH) is sustained. The stability of the cell's genetic information is maintained by the BRCA1 functions.
The repair of the damaged DNA involves a certain protein for which the instructions are provided by the BRCA1 gene. The BRCA1 protein reacts with numerous other proteins in the nucleus of many types of normal cells. These types of proteins which react with the BECA1 protein includes the BARD1 (BRCA1- associated ring domain protein 1) produced protein and the RAD51 (Recombinase A Deficient 51) genes. They react to repair the DNA mutations. Several factors can result in the mutations such as environmental, natural or medical radiation. Besides that the mutations can also be created when genetic material is exchanged by the chromosomes in the process of cell division. The growth of embryos and regulation of genetic activity is carried out by the BRCA1 protein by reacting with numerous other proteins for the process of cell division. For instance it results in the formation of multi sub-unit protein composites such as the BRCA1 linked genome surveillance complex (BASC) 7. This is done by commuting the nuclear phosphoprotein which in turn creates combinations with DNA damage sensors, signal transducers and other sorts of tumor suppressors. The RNA polymerase II and histone deacetylase complexes links with this encoded genes through the C-terminal domain. The DNA repairing of the double stranded breaks and transcriptions is primarily dependent on this protein complex.
Scientific researches have been able to identify about 850 of the clinical mutations. Among this majority of the mutations are held responsible for breast cancer. The mutations hinder the process of gene formation from one copy of the gene or they result in the production of BRCA1 proteins which are abnormally short in version1. Many of the studies have revealed that a BARC1 protein with a defect will have its abilities restricted in the repairing of the damaged DNA. The defects multiply further aggravating the problems leading to the cells to proliferate uncontrollably and create tumors. An abnormal protein is also formed as a result of these mutations which are normally found to change the amino acids to BRCA1 protein8.
The BRCA1 genetic mutation can commonly be detected through the PCR (polymerase chain reaction). In case of being aware of a certain mutation spectrum, a high throughput or a multiplexed mutation-specific technique is applied24. Only small amounts of DNA sample is required in the case of exponential amplification of a reaction, which is also simplified, cheap and extremely sensitive (assumption of 100% efficiency). One should be extra careful when performing the test to make sure that there is minimum contamination to optimize the results. The contaminations may cause amplification of the spurious DNA owing to its sensitive nature 24. The PCR settings have been optimized with the help of many protocols that have been developed for this purpose.
The regulation of the breast and ovarian epithelial cells is primarily dependent on the breast cancer susceptibility protein 1 (BRCA1), which is a tumor suppressor gene. The wild type allele contributing in the formation of cancer needs to be removed in the mutation of the BRCA1 found in women. These mutations are commonly found in the familial breast and ovarian cancer11. The research was done with the objective of finding out the optimal conditions required for the detection of BRCA1 gene with the help of PCR (polymerase chain reaction) and the agarose gel electrophoresis. The breast tissue DNA samples have the best option in the form of PCR, which is simple, quick and a cheap form of molecular technique. The DNA extraction process, the concentration of the template DNA and the PCR optimization can influence the detection of the amplifiable products 13
When constructing a PCR reaction of an unknown complex, a crucial and a time consuming procedure has to be applied, which is the optimization of the protocol. An adequate choice of the primer set is very necessary25. A primer designing software was used for the construction of specific BRCA1 primers (Forward/Reverse) which incorporated the exon 15 sequences to eradicate the complications that may arise owing to the primer-dimers formation and nonspecific product formation. The efficiency and the specificity of the PCR are greatly affected by the deigning pattern of the primers.
Success was achieved in the isolation of DNA from THP-1 and MCF-7 in the first experiment. The Chelex purified methods used for the DNA extraction gave results of satisfactory purity and quantity (Table 2). The method using Chelex is a good option for the PCR as it uses only one single tube for the extraction of DNA and eradicates the inhibitors of PCR. Therefore it effectively reduces the levels of contamination in the laboratory experiments. The range of concentration of DNA measured with the spectrophotometer was from 582 Î¼g/ml for MCF-7 to 1560 Î¼g/ml for THP-1. The standard that determines the DNA purity level is 260/280 nm absorbance ratio which shows the protein contamination.
This ideal ratio of 260/280 absorption is usually obtained from 1.8 to 2.0 which shows a satisfactory purity level in the DNA and minute protein contamination (Marteau)14. The absorbance ratio of 1.4 indicates poor DNA purity. The samples in this study indicated a good range of the levels of purity which was found to be from 1.60 for MCF-7 to 1.80 for THP-1. The pH and the ionic strength determine the absorbance of A260/A280 ratio. The A280 decreases with the increase in the pH while the A280 remains undisturbed. This means that the ratio A260/A280 increases. According to the ratio of 1:40, the DNA was diluted in water15. The ratio of A260/A280 was lowered owing to the acidic nature of water.
A buffer solution with a slightly alkaline pH should be used to ensure accuracy in the readings, such as TE (pH 8.0). By the multiplication of DNA concentration with the finalized purified sample volume, the total yield was obtained (see Appendix II). According to the results the MCF-7 and THP-1 yield was 47,127 Î¼g respectively. The agarose gel electrophoresis and UV spectrophotometry determined the purity, quality and concentration of the DNA samples. The agarose gel is useful, as it provides convenience in pouring it easily and it will not denature the samples. In addition the samples can also be recovered. The gel has a drawback as it can melt during the electrophoresis and it can exhaust the buffer. This will result in the genetic materials running in unpredictable forms30.
Columns packed with ionic exchange or silica based resins and matrices can also be used for the isolation of the DNA. An increased throughput of samples is enabled with the columns. It reduces the time utilized in isolation as compared to the traditional solvent based extraction. In addition it also provides an increased amount of the DNA recovered and it also enhances the quality of the purified DNA 15.
An optimum PCR template DNA for expressing Î²-Actin and BRCA1 was obtained in the second experiment. This was done by measuring the PCR band's intensity. The results indicated that the PCR's optimum template DNA for Î²-Actin was 100ng (see Figure 1, A). In the case of BRCA1 the DNA optimum template was 150ng (see Figure 1, B). Amplifications may be caused by an excessive concentration of DNA template while poor results or excessive cycle time can be the result of the desired gene being amplified in too little quantity25. The optimal concentration ranges should be verified through testing of various template concentrations covering orders of magnitudes at the outset of optimization 25. The false positives that may be caused by the contamination should be ruled out by the usage of negative controls. I product of Actin and 2 products of BRCA1 were created by the PCR. Non specific PCR products may be caused by the faults in the design of the primer, setting of the reaction at room temperature, suboptimal reaction conditions and the suboptimal thermal cycling conditions.
By measuring the intensity of PCR bands, the Î²-Actin and BRCA1 expression's optimum PCR annealing temperature were done in the third experiment. According to the results the 61.0ËšC was the optimum PCR annealing temperature for Î²-Actin primers and 65.2ËšC was the temperature for BRCA1 (see figure 2, B). The PCR product was indicated in lane 1 of BRCA1 gel because of the negative control. This may be the result of carry over contamination, cross- over contamination and faulty primer design. The same gel generated about 2/3 of the PCR products which were also due to the above mentioned issues in experiment two. The primer's melting temperature should determine the annealing temperatures and ideally the annealing temperatures should be below the lower melting temperature of the two primers16. The computer analysis determining the calculated primer's annealing temperatures which were differing from the optimal annealing temperatures. The presence of a template and a buffer system must be the reasons behind it as they affect the annealing temperatures of the primers; hence it is important to optimize the annealing temperatures.
By the measurement of the intensity of PCR bands, the maximum PCR MgCl2 for expression of Î²-Actin and BRCA1 were determined in the fourth experiment. This technique is highly beneficial for the magnesium ion concentration. 17 The stringency of the primer was decreased by increasing the concentration of the magnesium ion 26. Magnesium ions in low concentrations may result in declined efficiency of the reaction while the high concentrations of the ions may result in the declined specificity of the reaction. The calculation of the magnesium ions concentration should be done as a function of the concentration of the nucleotide 26. The testing of the magnesium concentration ranging between 0.5 to 4.0 mM above the total nucleotide concentration should be done for PCR protocols. According to the results the optimum PCR (MgCl2) for the Î²-Actin expression was found to be 2.0 mM. While 4.0 mM was the optimum PCR (MgCl2) for the BRCA1 expression.
In the fifth experiment, through measuring the PCR band's intensity, the optimum PCR number of cycles for the expression of Î²-Actin and BRCA1. The results indicated the Î²-Actin PCR cycle number to be 30 and 33 cycles (see figure 4, A). But in the case of BRCA1 there were no amplifications found which could be owing to the following reasons:
Degradation of DNA.
Degradation of misplacement of the PCR component.
Implementation of too less cycles.
Increased annealing temperatures.
Not enough templates present.
The sub optimal concentrations of Mg2+ and degradation of primers.
To calculate the PCR cycle range is the main purpose behind the optimum PCR cycle number in which a linear exponential phase denotes the intensity of the PCR bands. The growth curve of the PCR against the number of PCR cycles should be sigmodial (S shaped) when optimizing the PCR conditions. The curve should consist of three phases: log-linear phase (the exponential amplification phase), the lag phase and plateau (figure 8) 18.
Lag phase: it signifies the initial amplification cycle in which no product is detected.
Exponential phase: this phase denotes the period when the amplification efficiency is at its best and is constant over a period of time.
Plateau phase: the reaction plateaus when the amplification efficiency declines.
During the exponential phase, it is necessary that the PCR products are measured where a consistent relationship exists between the product amount and input DNA. To avoid the amplification of the background products, optimization of the number of PCR cycles is done.
By the measurement of the intensity of the PCR bands, the optimum PCR (Buffer, dNTPs & Primers) for expression of BRCA1 was done in the experiments 6, 7 and 8. According to the results the optimum PCR buffer, dNTPs and Primers was found to be 1.5 µL, (0.080 & 0.100 mM) & 0.2 mM respectively17. Buffering agents like the Tris-based buffers and salt like KC1 were used as buffers in many of the reactions. The DNA polymerase activity is affected by the regulation by the buffer of the pH of the reaction. The DNA activity of the polymerase will be increased by 50-60% by the modest amounts of KC1 as compared to the activities without the KC1. Empirical means should be employed to determine the concentrations of deoxynucleotide triphosphates (dNTPs) with the help of titration of Mg2+ concentrations. Lower specificity may be caused by high concentrations while poor yield of the product may be caused by low concentrations26. Mispriming and collection of nonspecific product may be caused by high concentrations of primer. High concentaryions may also result in an increased probability of template independent artefact termed primer dimer while unsatisfactory yield of the amplified product may be caused by low concentrations of the primer-dimer1.
Over the past 20 years, developments in the PCR have been introduced to meet the researcher's needs and the clinical molecular diagnostic labs.
Taq is prevented through a technique called as Hot start PCR20 , which may extend the primers till a temperature of 60-80 °C are obtained which are normally done by withholding Taq from the reactions till a temperature is reached. The Taq extension of primer dimmers or primers is prevented through this step, which have annealed to non specific DNA regions at low temperatures. This may be during the preparation of the reagent mix. When the target DNA is a small percentage of the total DNA, the result is the improved specificity and a PCR yield. In the reduction and elimination of the unwanted products, the Nested and seminested PCRs are often quite useful, simultaneously increasing the sensitivity19. The PCR optimization approach is represented by the Touchdown (TD) PCR19. Careful design of the multiple primers pairs and PCR optimization is required for the Multiplex PCR to avoid the formation of primer-dimers and other nonspecific PCR products that may hinder the specific product's amplification. To verify the presence of amplifiable nucleic acid in a sample, multiplex PCR is frequently used. An example may be the housekeeping genes amplified with the gene sequence. The detection of enterovirus and herpes simplex virus (HVS) nucleic acid in cerebrospinal fluid (CSF), analysis of multiple BRCA1 loci in breast cancer patient, detection of pathogenic enteric bacteria in stool, amplification of multide microsatellite loci for bone marrow engraftment analysis and identification of different bacteria in a respiratory infection 21 are the examples of multiplex PCR amplifications.
The following system may benefit the detection of BRCA1 gene or its mutations in the clinical samples. Exon 15 amplification in BRCA1 gene was not indicated in the previous studies. The transcription present in yeast and mammalian cells can be activated by the function of the C-terminal region constituting of exon 15-24 (aa 1560-1863) of BRCA1 fused to GAL4 DNA binding domain. The exon 15 was the most suitable exon for the amplification 28. More than 75% of the BRCA1 genetic mutations happen at the exons 2, 5, 11, 16, and 20, as stated by the Breast Cancer Information Core (BIC) 29. The technique used in the detection of the expression (RNA) of BRCA-1 gene, also known as reverse transcription-polymerase chain reaction (RT-PCR). The mRNA detection and quantitation employs the most sensitive technique called as RT-PCR.
The PCR is able to provide 100% sensitivity and specificity when used in molecular biology laboratories, used with accurately designed assays in a controlled environment. Although in majority of the cases the results are not according to the potential owing to the occurrence of issues such as ignoring the basic principles in assay design and optimization. The PCR performance is dependent on the key factors such as:
Selection of an adequate detection system.
Utilizing the software available for the best primer and probe design.
Analysis of sample quality and controlling inhibitors.
Abstaining from environmental contamination.
Optimization of concentration and reagent quality.
Modification of thermal cycling protocol for maximum sensitivity and specificity.
Many variables determine the specificity and efficiency of PCR such as MgCl2 concentration, primer concentration, annealing temperature, dNTPs, cell cycle and buffer concentration. The primers may also specify the parameters for optimum amplification. In order to maximize the yield obtained, the system needs to be optimized for individual primers and template.