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Analyzing a PCR Test Report Through Key Research Assignment Sample

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Analysis of PCR and Western Blot Test Results in Residential School Students Assignment Sample

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This assignment will discuss a crucial research topic which is the PCR test and western blot test done by residential school students. This assignment will analyze the test result of PCR test result and western blot test results which are found after doing the test. PCR tests mainly detect the genetic material of a specific type of organism. This test helps to amplify the sequence of the DNA. The PCR test, allows the researcher to create numerous copies of the small section of RNA or DNA (Zhou, et al, 2018). Western blot test help to identify and separate the proteins and nucleic acid. By this western blot method, the protein mixture is separated on the basis of its molecular weight, this process is done with the help of gel electrophoresis. After doing the test it is transferred into the membrane and produces a band of each protein molecule.

Background

The background of this research is to analyze the result of PCR and Western blot test results. PCR test help to detect the genetic materials of an organism, and amplify the DNA sequence of an organism. Western Blot test results separate and identify the nucleic acid and protein.

Aim

The aim of this research is to improve the knowledge about cell physiology and their system. This research helps to analyze data and interpretation. This research result critically interpret and synthesize the knowledge of cell physiology (Shirato, et al, 2020). It also helps to develop the skill for preparing the data in a scientific way. This research is held on residential school. This research analyzes the test result of PCR and Western Blot tests.

Hypothesis

H0- This research analyzes the genetic materials of an organism, and amplifies the DNA sequence of an organism.

H1- After doing this result it helps to create various small sections of DNA and RNA.

H0- To analyze the western blot test result, it helps to identify and separate the nucleic acid and protein.

H1- The protein mixture contains all the proteins which are associated with the particular tissue.

Materials and method

PCR (Polymerase Chain Reaction)

This method helps to analyze the short DNA or RNA sequence. PCR method is used to amplify the selected section of the RNA and DNA. Previously, the amplification of the DNA involved in the cloning of the DNA segments help to detect the bacteria’s expression, and this test took weeks but present-day This test is done in the tube and this test only takes a few hours. PCR uses the molecule that helps to copy The DNA sequence (Naji, et al, 2020). 2 primers are used in the “single-stranded DNA sequence” these two primers are synthesized and correspond to the beginning and end of the stretch DNA are copied. The enzyme called polymerase that moves along with the DNA help to read the code and assemble the copies of the DNA.

PCR process

3 steps are involved in the polymerase Chain Reaction. These steps repeat 30 or 40 cycles. These 3 cycles are automatically cycled and the user device is heated and cools rapidly. Each of the steps has 3 steps denaturation, annealing, and extension (Becherucci, et al, 2021). These 3 steps are done in 3 distinct temperatures.

Denaturation

In the denaturation step, at 94-degree temperature double-stranded DNA is melted and as a result, 2 single-stranded DNA are formed.

Annealing

The annealing method occurred 54-degree celsius. The anneal pair with the “single-stranded template”. The polymerase attaches and copies the template.

Extension

The extension step, this step occurs at 72-degree celsius. At this temperature polymerase works at the best level, and makes double-stranded DNA. 

The purpose of PCR test

This PCR test helps to study the evolution of human. Help to detect the clone of the DNA which help to recognize the biological relationship.

Western Blot Test

This test is also known as an antibody test. This test is done with the blood sample of the specimen. This test is used to confirm the result of Lyme disease or HIV test. This is also known as the protein immunoblot test (Gu, et al, 2019). This test helps to detect the “ribosomal RNA binding protein”. This test uses the procedure called gel electrophoresis and it helps to detect the protein by their molecular length and weight.

Procedure

This is a simple blood test. For this first extract of the protein, then gel electrophoresis occurs on the SDS page. Then blotting occurs in a capillary or electrical method. For blocking BSA, the method is used. Then treated with the primary antibody. After treatment with the primary antibody secondary antibody is used and then treated with the “specific substrate”.

In the residential school, pc3 and LnCaP cell lines are used. On day 1 no proliferation occurred. On Day 2 the residential school used pc3 and LnCaP cell lines (Gui, et al, 2020). They all used the PCR method. In this PCR method trpv1, b2m, TRPV6, GAPDH, TRPv6, GAPDH, trp1, b2m, AR, b2m. On day 4 pc3 and LnCaP cell lines are used. On Day 4 only the western methods are used.

This research study was held in a residential school. In this research 5 participants participated. For conducting this research they use PC3 and LnCap cells. On day 2, they did a PCR test with the cell PC3 and only 1 participant use LnCaP for the PCR test (Pan, et al, 2020). For conducting this research they use trpv1,b2m, TRPv6, GAPDH, and for LnCap cell they use AR, b2m as a primer. On day 4, all the participants were done with the western test for this test they use the PC3 cell line, and only one participant use the LnCap cell line for doing the Western test.

Result

Calcium Assay-LNCaP

In the calcium Assay, as vehicle control 12.55, 10.89, 12.45, 10.73 and 7.12 are used. The CAP 100uM are 177.54, 163.74, 175.5, 156.45 and 182.93 respectively. The value of the CPZ 100uM are 11, 9.34, 9.34, 8.6, 4.91. The sum of 100uM and CPZ 100uM value is 30.42, 29.6, 25.23, 32.85, 44.02. The total vehicle control is 10.748. The total CAP 100uM is 171.232. The total CPZ 100uM is 8.638 and total value of sum of 100uM and CPZ 100uM is 32.424. The mean value of vehicle control is 0.98. The mean of CAP 100uM is 4.85. The mean of CPZ 100uM is 1.01 and mean of (100uM + CPZ 100uM) is 3.14.

Analysis PCR test

In this PCR test “LNCaP” has been used. The first lane is for the DNA ladder. In 2 to 5 lanes β2M (House Keeping) has been used. It showed light-colored bands. In 8 to 11 lane AR(gene of interest) has been used and it showed no bands which means in the sample it is absent.

In this PCR test “LNCaP TRPV1” has been used and the light band has been noticed in between 6000bp to 10000bp approximately in 8th and 10th lane.

In this PCR test “LNCap TRPV6” has been used and light band has been noticed at approximate 6000bp to 10000bp. The band has been noticed in 2,4,8 th and 10th lane.

(Source: Provided)

From 200bp to 1031bp it shows double band and from 1500bp to 10000bp shows single band. Well length is 8cm and 10 microlitre sample is added within each cell.

 Western result analysis

The upper mentioned image use 150 Kbp marker. The well 1 VC intensity is prominent in 72. The well 2 VC intensity is prominent in 73 which is more prominent than the well 1 VC. In the well Cap the intensity is prominent in 72 . This band is very much prominent than two well VC. In the well VC 1 intensity is prominent in 49, which is very much prominent, and in the well cap intensity is prominent in the 49. They are very prominent band. The intensity is very prominent.

The upper mentioned image use use 150 marker, well 3 VC is prominent in 92. Well 4 VC is prominent in 92 and well 5 VC is prominent in 92. Well cap prominent in 92. Well cap prominent in 92 and well cap is prominent in 92. These well cap are very much prominent than well VC. well VC 3 is very prominent by using 150 marker. Well VC 4 is very prominent this is very intense and well VC 5 is also very intense the fragments of the DNA is more prominent. The well Cap1, in marker 37 is very prominent, and DNA fragments are more prominent the well cap 2, in marker 37 is very prominent and the intensity of the DNA fragments are very prominent in this well. In well cap 3, using 37 marker the intensity of the fragments are very prominent.

In case of LNCaP TRPV1 western, 150,100, 75, 50, 37 and 25 marker are used. Here in this picture marker 75 first VC is very light, 2nd VC is also light and Cap is also light. For 150 marker VC1, VC2 and Cap is not visible. In case of 100 marker VC1, VC2 and Cap no band is visible. In case of 75 marker VC1, VC2, Cap only VC 2 and Cap is visible. In case of 50 marker VC band is not visible, where as VC and Cap is visible and they are darker band than 1st VC. In case of 37 marker VC1, VC2, Cap no band is visible. In case of 25 marker VC1, VC2 and Cap no band are detected. The upper mentioned image use 150 Kbp marker. The well 1 VC intensity is prominent in 72. The well 2 VC intensity is prominent in 73 which is more prominent than the well 1 VC. In the well Cap the intensity is prominent in 72 . this band is very much prominent than two well VC. In the well VC 1 intensity is prominent in 49, which is very much prominent, and in the well cap intensity is prominent in the 49. They are very prominent band. The intensity is very prominent.

Discussion

After doing PCR and western blot tests it was analyzed that the PCR test help to detect the genetic materials of a specific organism, doing this test helps to amplify the DNA sequences of a particular organism. For conducting this research the participants use PC3 and LnCaP. There are 4 primers were used for conducting PCR tests that are trpv1, b2m, TRPV6, and GAPDH, when the participants use LnCaP for conducting PCR results they use AR, b2m as a primer.

When the student uses LnCaP as a cell line, In 2 to 5 lane β2M (House Keeping) has been used. It showed light-colored bands. In 8 to 11 lanes AR(gene of interest) has been used and it showed no bands which means the sample is absent. In this PCR test, LnCap TRPV6 has been used and a light band has been noticed at approximately 6000bp to 10000bp (Frank, et al, 2020). The band has been noticed in 2,4,8 th and 10th lanes. In the western blot analysis method, the band is also noticed by using the marker. This band sometimes forms darker and sometimes lighter. When the band shows darker it describes the intensity of the species. It helps to amplify the DNA of the organism, by the western blot method also helps to identify the protein and nucleic acid of an organism or species which are used for conducting this test result.

A critical analysis by using literature

According to Rådström, et al 2019, in the given research study they recognized polymerase chain reaction is a sensitive, rapid test and it helps to analyze the specific kind of nucleic acids. The kinetics and sensitivity are reduced when it is applied directly to biological samples like feces and blood. The pre-PCR procedure has been developed to remove the impact of the PCR inhibitor. This “pre-PCR processing” help to detect PCR products such as sampling the products, the preparation of the sample, and the amplification of the DNA. the objective of pre-PCR processing research is to turn a complex biological sample into the biological complex sample with the target cell or the nucleic acids into the PCR amplifiable samples through the amplification condition and sample preparation. There are several strategies used for the pre-PCR tests (Shirato, et al, 2020). The optimization of the amplification of the DNA through the use of alternative DNA polymerase or different kinds of amplification of DNA polymerase. Then optimize the method of the preparation of the sample. Then optimize the procedure of the sampling and then combinations of the various strategies. This review helps to define the different type of pre-PCR processing methods for the inhibition of the PCR and help to precise and accurate amplification of the DNA.

According to Samuel, et al, 2019, in the given experiment they detect the quantification of the protein by maintaining the approach of the rapid western blot method. In this method, certain proteins are used. Analysis of the western blot method is used widely. It helps to detect the target protein which is based on the specific kind of antibodies. This procedure is very much time-consuming. It reduces the processing. The challenges of the quantification of the reliable protein are used by the western blot technique. This method is used to normalize the adequate data, which is used in the western blot method, and detect the stability of the protein. Normalization of the protein is basically performed depending on housekeeping proteins like ß-actin and glyceraldehyde 3-phosphate dehydrogenase (Otsu, 2021). The assumption of these 2 proteins is constitutively expressed and across the same level of the experiments. There are numerous studies are used to make the suboptimal approach. Here use another strategy that is the normalization of the total amount of protein, where the target protein is used to relate to the total amount of protein. This normalization of the protein approach is independent and the reliability and quantification are increased. There are several western blotting methods are available like fluorescent detection, radioactive detection, chemiluminescent detection, and colorimetric detection. This conventional colorimetric staining suffers from low sensitivity. This approach is independent. In addition, increase the reliability and quantification. The “chemiluminescence- based methods” are the straightforward method, and help to detect the signal that does not correlate linearly to the abundance of the protein. .thses have a narrow protein dynamic range. The radioactive method brings to harm the human body. So that to get relief from this harmful method stain-free method is used (Nurfawaid, et al, 2021). This method is a combination of the stain-free fluorescence method and the standard fluorescent method. The rapid western blot protocol combines the fast blotting method by using fast immunostaining and the iBlot system method.

Limitation of PCR test

PCR test is use the limited diagnostic tool, this test is used culture test for antibiotics or drugs. The test result of PCR used in the treatment management, the result are interpreted by the professional with the history of the patient and their symptoms and clinical sign. There are in several case negative results are formed by the collection of the improper sample, the insufficient amount specimen, During storage and shipping degradation of the nucleic acid, due to test error., non-homogeneous distribution of the organism.

Limitation of the western blot test

The western blot test result is fail when the concentration of the protein is not in enough amount when the participants use the wrong sample buffer for conducting the test (Lachowicz-Wolak, et al, 2022). The concentration of the antibody when wrong. This is a time-consuming process. There are erroneous bands are formed, due to the insufficient transfer no bands are formed. The availability of the primary antibody is very crucial. This process is very delicate. Incorrect labeling of protein can happen due to the reaction of secondary antibodies. This western blot technique always requires a well-trained technique. For the western blot, the method protein must be denatured.

Conclusion

After doing PCR and Western blot test it is concluded that the PCR test basically occurred to amplify the DNA sequence of an organism. Western blot test help to detect and identify the protein and nucleic acid of the organism. Here this research study found different types of bands in the marker. The darker band means the higher intensity of the sample.

Reference

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