Common Questions On Ligation And Cloning In An Essay.

Why is it easier to ligate sticky ends then blunt ends?

1.Why is it easier to ligate sticky ends then blunt ends?

2.How many bases in a restriction site sequence need to be methylated to protect that site from restriction enzymes?

3.What does a DNA substrate require [or have to have] to be ligated?

4.What is the value of using phosphatase when preparing substrates for a ligation?

5.We do not use DNA ligase without ensuring we have first removed phosphatase from the reaction. Why??

6.The specific example of TOPO cloning given in class involved an additional technique called TA cloning where the T and the A refer to specific bases on the insert and/or vector. What is TA cloning and how is it used here?

7.Could TOPO cloning in theory or practice be done without using TA cloning?

8.What is one advantage  of cloning PCR fragments via the combined TA- and TOPO-cloning over the use of inserts isolated using restriction fragments?

9.Imagine you were given a mixture of a single plasmid but some of that plasmid had been cut in one spot by a restriction enzyme, some had been nicked in one strand, and some had not been touched at all and so was supercoiled just like when it came from the bacterium. Would these run differently on an agarose gel, or would some or all run at the same position?  Explain your answer.

10.Name two ways that a recombinant clone can be put into a bacterial cell from a ligation reaction tube?

11.What is meant by the term “vector” as it relates to this course?

12.What is a gene library?

13.Why do we make libraries?

14.In the various examples I went over in class, would each clone in a genomic library consist of 1 gene, or more than 1, on the average?

15.Why would anyone worry about the “p” value (the probability of success) when calculating how many clones would be needed to have a “good library”?

16.What is the advantage of making a genomic library from partially digested DNA?

17.If small clones are easier to transform, why don’t we ensure that all inserts are <500 bp?

18.What parameters can we vary to ensure that we get a partial digest?

19.Within the context of this course, what is a label?

20.What are the roles of the tra protein? [be prepared to speak about 2 very different, although consecutively operating, roles.]

21.How does replication from oriv differ from replication from orit?

22.“Why” do plasmids use two dfferernt modes of replication [that is, why can’t they do everything with one?]?

23.How does the presence of antibiotic resistances, or novel metabolic pathways, ensure plasmids or transposons are retained in their hosts?

24.Are these antibiotic resistances, or novel metabolic pathways, beneficial or wasteful when cells are grown in complete media without unnecessary chemicals or antibiotics?

25.Within the field of genetics, what is the meaning of complementation?

26.What makes transposons so useful?

27.In the field of genetics, what is a “footprint”?

28.Why did we use a narrow host range plasmid to introduce a transposon into Myxococus?

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.

How many bases in a restriction site sequence need to be methylated to protect that site from restriction enzymes?

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.

What does a DNA substrate require [or have to have] to be ligated?

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.