1. In your own words explain why this paper is important or significant
The article is very informative and significant since it introduces another breakthrough in DNA sequencing and analysis. It highlights the polymerase chain reaction which is the new way of identifying mutant genes in both homozygous and heterozygous organisms. The technique uses primers to amplify segments of the DNA by many folds such that it clearly identifies the mutant genes faster than all the other methods. The paper points out important milestones that genomics and gene sequencing has undergone over the years. Geneticists usually use the cloning method of sequencing which takes up to three days before results were identified and greatly hampered the process. In the cloning process, scientists had a hard time preparing the laboratories since it was a delicate process. In addition, if the genome was from a heterozygous multiple studies and clones the previously mentioned process had to be done to distinguish the DNA sequences.
The article also documents different methods applied in the identification of different alleles other than the cloning method. One is the hybridization method where a mutant can be fully located in DNA with short segments especially if the nature of the mutation is clearly known. Another method highlighted is the ribonuclease cleavages where RNA is used to identify changes in the DNA bases and a comparison of RNA in the genomes is done to detect any mismatches. Other approaches used in the detection of mutants are the denaturing gradient gel electrophoresis. Despite the advantages of all these methods, the piece makes it clear that there exists some difficulty in locating the mutant site. The mentioned notion is owing to the reason that the methods have significant errors and assumptions. It gives us a chronology of processes undertaken in justifying the Polymerase Chain Reaction (PCR) over the others. In addition, it clearly documents the laboratory procedures which can be referred to in any further studies to be conducted.
2. State which organism(s) and/or cell types were being studied
Organisms being studied in the article are of human beings which are used for laboratory analysis. Therefore, it is crucial to note that Escherichia Coli is used in the experiment. The new DNA sequencing of PCR has been successfully implemented in the human mitochondrion genome. The amplification of the DNA has been done to the genome to identify mutant alleles. The primary objective of the study was to identify and highlight different DNA sequencing methods precisely the PCR method. How the method will work in the identification of the mutant alleles in humans so as to reduce the hectic way of the cloning process.
3. List each experiment conducted and give a brief description of the techniques employed for each
Several experiments were conducted in the study and one such is:
- DNA implications: it uses the Saiki method but with some minor variations made to the process. For instance, after denaturation and centrifugation, the samples are incubated in at room temperature in a water bath before the addition of the virus (E. coli).
- DNA sequencing reaction: it involves recovering of the reaction by use ethanol and termination sequence of Dideoxynucleotide.
The results of the studies between segments of the DNA, base changes, were identified, and a bigger number of mutations have been known to lead to hemoglobinopathies. Based on Klenow fragment, the PCR amplification and AMV transcriptase continue to increase steadily as the DNA signal weakens. The difference in the results is not known and should be investigated. The notion is due to the complex structure of the genome and the DNA. The results from the study are based on the DNA amplification. In addition, it reflects on the experiment that clearly distinguishes different DNA sequences and takes a short period in determining it. Despite all the merits of the Polymerase Chain Reaction Method (PCR) sequencing of the genome DNA is lengthy and complex process which is dynamic.
The article highlights different methods of DNA sequencing and their merits and demerits. The agenda of the article was to portray and document the new methods of human genomic sequencing and specifically the PCR (Polymerase Chain Reaction). The method is stated to have several advantages over the cloning process method. Some of the advantages include the ability of the method to produce results in a faster and more reliable. It gives it an upper hand over the cloning process which takes several weeks of preparation and production of only homozygous results. Various experiments are done such the DNA amplification method and DNA sequencing reactions to establish the most reliable way of distinguishing mutant genes. The results indicate the difference in the experiments because as the DNA was being amplified, the DNA signal became weaker and weaker. It however leaves for investigation to be done to ascertain the difference in the experiments.
4. An Experiment That Would Build This Paper
Lymphoblastoid cells 15% polyacrylamide/8M urea gels
Endonuclease HindIII DNA synthesizer
Oligodeoxyribonucleotide Deoxyribonucleotides and dideoxyribonuclotides
ATP Proteinase K
T4 Polynucleotide kinase Avian Myeloblastosis Virus(AMV)-to act as reverse transcriptase
Escherichia coli- to prepare the DNA polymerase.
Human DNA was the main specimen prepared from blood leukocytes and lymphocyte cells.
Restriction of endonuclease was done before amplification.
DNA amplifications were done by the method of Saiki et al with fewer modifications. Use of Hepes rather than Tris buffer assisted maintains the integrity of the DNA. After the denaturation and centrifugation steps, the sample was incubated only 20sec in room temperature water bath before adding of the large fragment of E.coli DNA polymeraseI.AMV was added for reverse transcriptase and incubated. The DNA was recovered through precipitation and subjected to electrophoresis in polyacrlamide sequencing gels.