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PCR Process and Types

Discuss about the Developmental Biology and Pathology for Cancer Research.

PCR means that Polymerase chain reaction and is widely applied in molecular biology and cell biology. [1]

This method is used to create several copies of DNA and it makes it possible to visualize DNA by the use of a dye known as ethidium bromide upon carrying out gel electrophoresis. This process occurs in a number of steps where deoxynucleotides, DNA, primers, and the polymerase enzymes are all placed in test-tube. This reaction involves a number of processes that include; first id to denature the double stranded DNA molecules by heating them at 96 degrees to separate them into two distinct strands. The determination of the melting point for heating the DNA determines the effects of the DNA bases namely A, T, C, and G to the primers used. The next step is annealing where the primers are added to the DNA strands and this occurs at a temperature of between 37 to 65 degrees Celsius. Then extension through a number of cycles is repeated in order to have so many copies of DNA. There are several types of PCR such as reverse transcriptase (RT-PCR), quantitative PCR (qPCR), and real time PCR. The RT PCR allows the researchers to determine the amount of DNA which is being replicates hence commonly used to amplify the messenger RNA. This method is quite crucial for the molecular biology in that it offers high speed due to the modern machines like the thermocyclers which have been invented. Using this machine, the PCR process can be completed within hours’ time as opposed to the cloning process which would rather take several days to complete. Uses: the PCR protocol can be applied for various activities such as genotyping, cloning, detection of mutations, forensics research, testing for paternity, and in the treatment of some genetic health conditions.

  1. Monosomy is the absence of one member of a pair of chromosomes. this means that in monosomy there are about 45 chromosomes in every body cell as opposed to the usual 46 chromosomes.in monosomy X, or Turner syndrome is a condition whereby a child is born with just one X sex chromosomes instead of the usual complete pair which exist as either two X chromosomes or as one X and one Y chromosomes. [2]
  2. Since the white and gray mice are all from pure lines, there are equal chances that the white and gray mice can be formed at equal probabilities. However, when the offspring’s are crossed with gray mice, there are high chances that the offspring’s of the second generation will have a high likelihood of the gray mice being formed. This is because in the second generation, there are more mice with gray characteristics which have been crossed with a pure gray mice population. This indicates that the genes for gray color are dominant and hence easily expressed.

Genotypes of children:  Ww, Ww, ww and ww (all gray).

  1. it receives the three codons which codes for one amino acid.
  2. the mRNA is added so many Adenine molecules on the tail. Again the GTP molecules are added to the head through capping.
  3. six. amino acid sequence will be: gly, tyr, gly, pro, gly, meth, pro, pro, meth, trpt, leuc, leuc, pro
  4. through regulating the stability of the mRNA and through regulating the rate of transcription.
  5. Trisomy is a chromosomal disorder which causes some birth defects. In trisomy, a person is born with an extra chromosome, whereby an individual has three as opposed to the usual pair. For instance, in trisomy X also known as Downs syndrome occurs when a child is born with three chromosomes 21 chromosomes. On the other hand, trisomy 18 is the abnormality whereby a person is born with three chromosomes 18 chromosomes instead of the usual pair. In the trisomy and monosomy abnormalities, the resulting individuals have birth defects. Under normal circumstances, a person is born with twenty-three chromosome pairs, that is, a total of forty-six chromosomes with each pair inherited from each parent. Therefore, in the case of chromosomal disorders like monosomy and trisomy, a person contains forty-five and forty-six chromosomes respectively.
  6. Inducible genes are genes whose expression is responsive to either changes in the environment or on the position of the cell cycle. An example of the common inducible genes is the Lac operon which harbors information concerning the information needed to synthesize various enzymes that are needed to break down lactose, a disaccharide [3]. Since this sugar is a disaccharide, it needs to be broken down into galactose and glucose for metabolism to provide energy. It happens that by essence, the bacterial cells prefer glucose as the staring raw material to make energy, as opposed to galactose. This is because glucose requires less amount of energy to metabolize glucose that does the galactose. One common system used by the inducible genes in some worms is the heat shock factor 1. In this system, the worms require a mild heating in order for the expression of the inducible genes to be activated.


On the other hand, constitutive genes are genes which are transcribed in a continuous manner which is the reverse of the facultative genes. More specifically, the expression of constitutive genes occurs when there is a close interaction between the promoter and the RNA polymerase enzyme without the need for an extra form of regulation. Their continuous expression is an indication that these genes code for gene products that play very important roles in the cell such as the maintenance of basic functions or structure.

  1. DNA gyrase- this is an enzyme which plays vital functions in the ATP dependent negative supercoiling process of the circular double stranded DNA. Broadly, the DNA gyrase enzyme belongs to the family of topoisomerases enzymes. This enzyme also plays vital roles in the creation of positive super coils. The roles of DNA gyrase occur through looping the template in order to create a cross, followed by cutting of the double helicases, passing another helicase before it and the releasing the break.
  2. DNA helicase- these are enzymes that involved in the unpack aging of genes in the cells. Since they are motor, the helicases can move along the phosphodiester backbone of a nucleic acid and separate the two annealed DNA or RNA strands apart. However, the separation roles of this helicase require the input of energy from ATP.

Role of DNA Polymerases

DNA polymerases- is a class of enzymes that are involves in the synthesis of DNA molecules from the basic units of individual deoxyribonucleotides, which are chiefly the building blocks of nucleic acids. More specifically, the DNA polymerase enzymes are involved in the replication and repair works in the DNA molecules. In the end, these enzymes create two identical DNA molecules from a single DNA molecule since they normally work in pairs. In this process, this enzyme reads the sequence of the original DNA molecule in order to make it possible to form two new strands which are identical. These enzymes, that is, DNA gyrase, DNA helicase and DNA polymerase are different but belong together because they are all topoisomerase enzymes. In this case, they play important roles in controlling the topological transitions of the DNA molecules.

The RNA refers to the ribonucleic acids which have a ribose sugar on their backbone. The different types of RNA include;


mRNA (Messenger RNA)- this RNA encodes the amino acid sequences of the protein polypeptide chains. This is the most heterogeneous type of RNA I terms of size and sequence as well. It carries with it the genetic information which has been copied from the DNA in the process of transcription in the form of triplets or codons which have three amino acids each. In this case, every codon specifies a given type of amino acid., although the same amino acid can be coded by several other different codons. In order to play its roles, the mRNA contains a capping on the 5’ end with the guanosine triphosphate. This capping enables the mRNA to recognize well in the process of translation and transcription. Additionally, the 3’ end of the mRNA has a poly A tail, meaning that it has several adenine molecules to prevent it from undergoing enzymatic degradation.

tRNA (Transfer RNA)- these performs the roles of bringing the amino acids to the ribosomes, which are structures where the protein synthesis takes place during the process of translation. These are the smallest types of RNA which play crucial roles in protein synthesis. Each of the twenty known amino acids has its specific transfer RNA which joins to it and transfers it to the elongating long stretch of the polypeptide chains. For their efficiency, the tRNAs have a clover leaf structure to make it strong enough by forming hydrogen bonds between individual nucleotide molecules [4].

Types of RNA and Their Roles

rRNA (ribosomal RNA)- these RNA works together with the ribosomes in order to facilitate the process of protein synthesis. The rRNAs combines with the cytoplasm to make the ribosomes. Theme structures move together with the mRNAs in order to facilitate the arrangement of amino acids to form long polypeptide chains. Moreover, the rRNAs also binds to the tRNA intruder to enhance protein synthesis.

snRNA (small nuclear RNA)- these work together with protein molecules in order to make complexes which are used to process the RNA molecules especially in the eukaryotes since they are absent in the prokaryotes.

Mitosis is the multiplication of the somatic cells in order to form two identical daughter cells for each of the cells. This process takes place in about five steps namely; prophase, prometaphase, metaphase, anaphase, and telophase.

During prophase, the chromatin material condenses making the chromosomes thick enough to an extent of making visualization easy. Then the centromeres separate and moves to the opposite sides of the nucleus. The centrosomes form so that the sister chromatids cans separate to form the spindle fibers.

In the prometaphase, the centrioles are located on the cell poles and the chromosomes assemble in the middle of the cell. The spindle fibers then get attached to the chromosomes and this is regarded as the shortest phase of the mitotic division.

During anaphase, each of the centromeres splits to release the chromatids free. The individual chromatids migrate to the cell poles and the cells starts to elongate. then the telophase stage starts whereby a nuclear membrane is formed. The short chromosomes also start to elongate to form long chromatins. A cleavage furrow is then formed, and this is a shallow groove around the cell plate. Finally, cytokinesis ensues and the cytoplasm divides to form two identical daughter cells.

Meiosis is the type of cell division I which there are two successive stages of cell division; that is meiosis I and II. This type of cells division is special because it involves the formation of sperms and eggs containing the required number of chromosomes. During fertilization, the sperms and eggs come together and fuse and thus there needs to be correct division and chromosome numbers to avoid birth defects. In meiosis 1, the chromosomes contained in the diploid cells separate to form four chromosomes in the daughter cells. On the other hand, meiosis II, takes place in a similar fashion to mitotic cell division. In prophase I, the chromosomes pair forms synapses through the formation of a chiasmata. This bivalent contains two chromosomes and four chromatids since one chromosomes comes from each parent cell.


In metaphase I, each of the two chromosomes aligns on the metaphase plate such that each parental homologue is on either sides. This gives a high chance for each of the daughter cells to receive either the father’s or the mother’s homologue chromosomes. During anaphase I, the microtubules become so short hence pulling the homologous chromosome pairs from each other. In telophase I, the chromosomes get to the end the cell divides to form two haploid cells. In this stage, the membrane of the nucleus forms back, and the chromosomes uncoil to form the chromatids. In prophase II, the nuclear membrane breaks apart and the centrioles moves to the opposite ends of the cell known as the poles. At the same time, the chromosomes condense together. In metaphase II, the chromosomes are arranged on the at the plate but not in pairs, but rather independently. During anaphase II, the sister chromatids are pulled to the opposite directions of the cell. Finally, in telophase II, the cells are broken down with the reappearance of the nuclear membrane. The chromosomes uncoil and four haploid daughter cells are formed. Moreover, in females, three polar bodies are formed (which are eventually destroyed) and one ootid or the ovum is formed.

10 each of the loci phenotypes has a frequency of 25%.

  1. By being universal, this means that the genetic code is preserved throughout the generation. Therefore, if there are changes to the genes, they are too minor, but the original code is constant.
  2. The population is not in the handy-weinberg equilibrium.

The frequencies are 25%, 25% and 50% for red, pink and white phenotypes respectively.

The observed genotypes are: RR, PP and WW

The expected genotypes are RP, RP and WW.

  1. The daughter will not be color blind but she will be a carrier of the trait.
  2. Parents genotypes: Xx and Yy

Children’s genotypes: XY, Xy, xY and xy. 

References

  1. Saiki, R. K., S. Scharf, F. Faloona, K. Mullis, G. T. Hoorn, and N. Arnheim. "Polymerase chain reaction." Science 230 (1985): 1350-1354.
  2. Gropp, A., B. Putz, and U. Zimmermann. "Autosomal monosomy and trisomy causing developmental failure." In Developmental biology and pathology, pp. 177-192. Springer, Berlin, Heidelberg, 1976.
  3. Baylin, S. B. (2016). Jacob, Monod, the Lac Operon, and the PaJaMa Experiment—Gene Expression Circuitry Changing the Face of Cancer Research. Cancer research, 76(8), 2060-2062.
  4. Ikemura, T. (1985). Codon usage and tRNA content in unicellular and multicellular organisms. Molecular biology and evolution, 2(1), 13-34.
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