1.The sticky ends consist of regions of unpaired nucleotides called overhang which readily pairs with another sticky end with a complementary overhang whereas the blunt ends have their nucleotides already paired between the two strands of the DNA hence cannot be easily ligated with other nucleotides (Pedersen et al., 2014).
2.Only two, that is, Cytosine and Adenine of the four base pairs of the DNA can be methylated therefore enough to repress restriction. The methylation of cytosine is common in both eukaryotes and prokaryotes and differs among species while adenine methylation is common in bacteria, plants, and mammals (Wu et al., 2016).
3.A DNA substrate requires four deoxyribonucleotide triphosphates (dNTPs) which are; dATP, dGTP, dTTP as well as dCTP which are cleaved forming deoxynucleotide monophosphates that get inserted into a new DNA strand. Also, the substrate requires ribonucleoside triphosphates (NTPs) which initiate and sustain the synthesis of DNA by synthesizing RNA primers and ATP. The ATP provides energy for the activation of the enzymes at the replication fork.
4.The process of sealing the nicks between adjacent DNA residues of a single-stranded break on the double strand substrate is done by the formation of phosphodiester bonds between 3’ hydroxyl and 5’ phosphate residues. Therefore, phosphates are important in the formation of the insert and plasmid to avoid blunt end ligating into the vectors.
5.It’s essential to remove phosphates to effectively inactivate the 5’ phosphate group to prevent self-ligation of the DNA using alkaline phosphatase enzyme for easy manipulation into the desired DNA prior to re-ligation.
6.TA (thymine and adenine) cloning is a simple convenient procedure for subcloning the PCR products through the amplification of Taq polymerase. In this type of cloning, both thymine and adenine have the ability to hybridize on separate DNA fragments in the presence of ligase enzymes. Therefore, they become ligated without the use of restriction enzymes.
7.TOPO cloning is feasible both in theory and practice without using the TA cloning method which does not use restriction enzymes. However, some restriction enzymes such as Phusion can be used to form blunt end TOPO vectors (Fontes, 2013).
8.In cases for the absence of viable restriction sites cloning is feasible through TA and TOPO cloning which produces linearized vectors making the procedure simple and faster.
9.The mixture can be run in a single gel electrophoresis at the same position. The DNA molecules are separated by the pores on the gel electrophoresis in respect to their size and shape whereby the smaller molecules navigate faster in the pores than the large ones. So, for the case of the mixture plasmid the DNA molecules will separate with the following decreasing rates; the supercoiled DNA, the linear cut DNA and finally the nicked circles.
10.The recombinant clone can be put into a bacterial cell through the process of transformation and electroporation.
11.A vector is a DNA molecule used in the artificial transfer of foreign genetic material from one cell to another where it is replicated or expressed. Essentially, there are four types of vectors; the plasmids, artificial chromosomes, viral vectors, and the cosmids which contain the origin of replication, a selectable marker, and sites for cloning. Vectors with the foreign genetic material are called recombinant DNA. These vectors are vital to genetics and other fields such as molecular biology as they are essential in the reproduction of the recombinant DNA for genetic engineering purposes.
12.The large collection of fragments of DNA cloned from a particular organism, tissue, organ or cell type which may contain an entire genomic sequence or the complementary DNA sequences of the messenger RNA contained in identical vectors constitutes a gene library.
13.The collection of the different DNA molecules from an organism cloned into a vector enhances the purification of the genes, its storage, and analysis. The library, for example, the genomic library aids in the identification on the novel genes of pharmaceutical significance, identification of the new gene that could have been silent in the host organism and in the deciphering of the complexity of the genomes. Also, the library serves as the source for genomic sequences for the transgenic organisms. They are widely used in the study of the functioning of the regulatory genes in vitro and cancer tissues as well as the creation of a cDNA library that helps in the determination of the genes that are expressed at a given time.
14.The genomic library would contain one gene that is, the whole nuclear DNA of organisms which include the gene exons and introns, the gene promoter, intragenic molecule and the origins of replication.
15.The p-value is important for it gives information on the approximate number of viable clones of infectious viral particles that were successfully created. In the subsequent experiments, these clones must contain the desired and favorable qualities to ensure they are efficient in genetic engineering.
16.In some cases, the fragmented DNA as a result of complete digestion by the restriction enzymes produces DNA molecules that are not intact. Partial digestion of the DNA fragments with a cutting enzyme such as Sau3A to produce a random collection of fragments of sizable distribution facilitating the formation of appropriate sized inserts.
17.The other important factor to consider in the preparation of the inserts is the ratio of the molar concentration of the insert to that of the vector DNA which should be about two for example 1:1, 2: 1 and 3:1. Therefore very small inserts affect concentration hence the ligation efficiency which in turn becomes very low.
18.For a partial digestion, the time of the reaction has to be modulated whereby the aliquots are taken at varying time intervals before the process is completed. The analysis of the aliquots gives the most appropriate temperature or condition that produces the desired quantity of the DNA fragment.
19.A label is a fluorescent tag which is a molecule that is chemically attached to a biomolecule such as an amino acid which facilitates the labeling and detection of the biomolecules. These labels utilize active derivatives of fluorescent molecules known as a fluorophore, for example, Ethidium bromide which selectively binds to a specific functional group of the molecule.
20.The tra proteins contain transfer genes (tra genes) which function in the non-sexual transfer of genetic content in bacteria. They also, encode for proteins that function in propagation. This enables the host cell to find a compatible donor to facilitate mating.
21.The oriT replication results to the transfer of DNA from host bacteria to recipient in conjugation and it is cis-acting and it gets transferred along with the DNA being conjugated. On the other hand, the oriV replication is associated with the prokaryotic and eukaryotic organism and it proceeds in bidirectional of unidirectional way.
22.The plasmids can replicate on their own since they possess replication origins. However, they can act as episomes whereby they reversibly integrate into bacterial chromosomes, therefore, using the bacterial replication mechanism. For this reason, they first have to replicate their own genetic material and later enhance replication in the bacteria for the purpose of conjugation
23.All the plasmids have antibiotic resistance in stress conditions such as the presence of an antibiotic the plasmid replicates its DNA and confers the host with resistance. The bacteria require the plasmid for survival although it deprives the bacteria of energy as it replicates. The role of the plasmid is vital that the bacteria cannot survive without it. Also, the plasmids carry genes that synthesis long-lived poison with a short-lived antidote for the bacteria which means the bacteria die in case the plasmids are lost.
24.Besides antibiotic resistance, the plasmids are vital in the reproduction of the host bacteria for they enhance conjugation and transfer of non-sexual genes between compatible cells. They also help the bacteria to survive in other adverse conditions besides antibiotics. Therefore, they are important for the bacteria with or without antibiotics in their environment.
25.Complementation is the phenomenon whereby two different species of organisms with similar homozygous recessive mutant are mated or crossed to produce an offspring with a wild-type phenotype. For complementation to occur the mutations must be in different genes.
26.The transposons are used in the genetic research as well as genetic engineering as vectors for removal and integration of genetic sequences. They are effective for insertional mutagenesis for they are semi-parasitic DNA molecules with the ability to replicate and spread in the genome of the host. For this reason, they are utilized in analyzing protein and gene function.
27.A genetic footprint is a method of investigating the specificity of binding proteins of the DNA in vitro. With this method, the interaction of proteins both inside and outside the cell can be investigated.
28. The narrow host range plasmid was used to introduce the transposon into Myxococcus using phage P1 for its ability to insert the genetic material at very many sites. The transposon effectively inserts itself into the bacteria’s genetic genome becoming part of the recipient’s chromosome. Upon insertion, to the Myxococcus it still remains in the host organism whereby it will continue to replicate (Cusick, Hager & Gill, 2015).
References
Cusick, J. K., Hager, E., & Gill, R. E. (2015). Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus. FEMS Microbiol Lett, 362, 1-8.
Fontes, A. (2013). Cloning technologies. In Pluripotent Stem Cells (pp. 253-261). Humana Press, Totowa, NJ.
Pedersen, R., Marchi, A. N., Majikes, J., Nash, J. A., Estrich, N. A., Courson, D. S., … & LaBean, T. H. (2014). Properties of DNA. In Handbook of Nanomaterials properties (pp. 1125-1157). Springer, Berlin, Heidelberg.
Wu, T. P., Wang, T., Seetin, M. G., Lai, Y., Zhu, S., Lin, K., … & Tackett, A. (2016). DNA methylation on N 6-adenine in mammalian embryonic stem cells. Nature, 532(7599), 329.
Essay Writing Service Features
Our Experience
No matter how complex your assignment is, we can find the right professional for your specific task. Contact Essay is an essay writing company that hires only the smartest minds to help you with your projects. Our expertise allows us to provide students with high-quality academic writing, editing & proofreading services.
Free Features
Free revision policy
$10Free bibliography & reference
$8Free title page
$8Free formatting
$8How Our Essay Writing Service Works
First, you will need to complete an order form. It's not difficult but, in case there is anything you find not to be clear, you may always call us so that we can guide you through it. On the order form, you will need to include some basic information concerning your order: subject, topic, number of pages, etc. We also encourage our clients to upload any relevant information or sources that will help.
Complete the order form
Once we have all the information and instructions that we need, we select the most suitable writer for your assignment. While everything seems to be clear, the writer, who has complete knowledge of the subject, may need clarification from you. It is at that point that you would receive a call or email from us.
Writer’s assignment
As soon as the writer has finished, it will be delivered both to the website and to your email address so that you will not miss it. If your deadline is close at hand, we will place a call to you to make sure that you receive the paper on time.
Completing the order and download