• Join over 1.2 million students every month
  • Accelerate your learning by 29%
  • Unlimited access from just £6.99 per month

You are given a cloned gene from Homo sapiens and are asked to identify structurally similar genes (potential orthologues) in a. Arabidopsis. b. Bombyx mori (silk moth).  How will you determine the expression pattern of the gene?

Extracts from this document...

Introduction

You are given a cloned gene from Homo sapiens and are asked to identify structurally similar genes (potential orthologues) in a. Arabidopsis. b. Bombyx mori (silk moth). How will you determine the expression pattern of the gene? Before analysis of related genes can begin it is important to extract as much information about or cloned gene is possible, as this will aid us in our search for potential orthologues. Firstly I am assuming that the cloned gene is derived from a DNA library and is contained within a vector as part of recombinant DNA. Knowledge about the type of library from which it originated is helpful (either cDNA or Chromosomal) for analysis of the sequence but I will examine that later. The first step is to determine the DNA sequence of this cloned gene. Assuming that I have been given only one colony of the bacteria containing the cloned gene for good experimental technique it is necessary to increase the amount of bacteria and so copies of the gene, before starting analysis. I would do this by setting up a liquid phase culture (LB for example) and incubating the colony I have been given until I have an appropriately sized quantity. In order to start analysis I must first extract the cloned gene from the bacteria and the plasmid vector to which it is attached. I would start by separating the components of the cell by size, in order to get rid of cellular debris. Initially I will have to break apart the cell to gain access to the plasmids, done by the addition of Lysozyme and EDTA. Because the plasmids used are of a lower weight than the rest of the cell contents after centrifuging they will stay in the supernatant whilst the rest will form a pellet at the bottom. See diagram. There is now a solution containing many types of damaged plasmids and small strands of DNA, which will affect the quality of our sequencing unless properly removed. ...read more.

Middle

All you have to do to search for an orthologue is to go to a website, e.g. http://www.arabidopsis.org/blast/ and use the BLAST program. Then simply type in your sequence hit search and any matches within the database are displayed graphically and then with a more informative text output telling you the percentage accuracy of the match and the gene it is matched to. Here is an example of a match I performed earlier just using some randomly typed in DNA sequence. Bombyx mori (silk moth) The search for potential orthologues becomes increasingly more difficult in the silk moth because unlike the Aribidopsis it is not yet fully sequenced. However you can still use the partially completed database to search at http://210.145.41.132/cgi-bin/lib_proc_MX . However when I tried using the same random sequence above it returned no matches at all and only 18 thousand sequences had been determined so far. Because I cannot simply BLAST the sequence I have to search for any orthologues manually. This can be done in two methods, depending on whether the gene is expressed frequently enough to detect in the silk moth or not at all. To determine whether the gene is expressed I would take a number of samples of tissue from the moth (muscle, neuronal) and then using the protein derived from the human gene as a template to would check for that protein by ding a biochemical analysis of the gene products. This would only be a quick search as the chances are that the gene in question is not expressed or is so rare that I will not be able to detect it. If however I do manage to find that the gene in question does have an orthologue that is expressed I can then move onto trying to ascertain the DNA sequence from the mRNA transcript and studying the similarities. Firstly I would have to isolate all the mRNA from the cell. ...read more.

Conclusion

Studying the translation product of the cloned gene can give us information as to how large the product is and how to look for it using other techniques. Hybrid-release translation (HRT) is used to create gene products using cell free translation systems. The gene products from the mRNA are created in vitro and are usually labelled through a particular amino acid. Which can then be run on a gel with another protein ladder and the size of the gene product can be determined. With this method it would therefore be possible to find out the absorbance of the product at a particular wavelength. The cells could be lysed and proteins purified and run it through an HPLC column attached to a UV detector (or mass spec if exact weight could be calculated) to see how much of the particular protein was being expressed. You could also use a GC but I think the protein would be too big for the coils and clog them up. The only other way I can think of to determine the gene expression pattern of the different organisms is to use antibodies. These would be created by challenging a rabbit with the protein in question. The rabbit would be left for a couple of days to create antibodies in an immunological response to the foreign protein. Then the antibodies in the blood could be extracted and have a luminescent marker attached, i.e. horseradish peroxidase. Now when the cells are plated out and exposed to the antibody if the protein in question if being expressed then they will bind to the protein. If the cells are then exposed to luminol the cells expressing the gene light up due to the chemiluminescence with the modified antibody. All the above methods are ways of detecting the gene products and hence expression patterns of the organisms in question. I have not mentioned the use of deletion sequences to determine the promoters and activators of the genes because in eukaryotes there is usually fatal damage done to the embryo if these sequences are disrupted, much more than prokaryotes. ...read more.

The above preview is unformatted text

This student written piece of work is one of many that can be found in our AS and A Level Genetics, Evolution & Biodiversity section.

Found what you're looking for?

  • Start learning 29% faster today
  • 150,000+ documents available
  • Just £6.99 a month

Not the one? Search for your essay title...
  • Join over 1.2 million students every month
  • Accelerate your learning by 29%
  • Unlimited access from just £6.99 per month

See related essaysSee related essays

Related AS and A Level Genetics, Evolution & Biodiversity essays

  1. Marked by a teacher

    An Investigation into the Mitotic Nuclear Division of Allium Sativum Root Tip Cells, and ...

    5 star(s)

    It is possible that the sharpness of the scalpel and mounted needle could be responsible for damage to those conducting the experiment, due to possible slippage or accidental movement when handling equipment. All the relevant cutting equipment should be handled with care and responsibility throughout the investigation.

  2. Regulation of gene expression in prokaryotes and eukaryotes.

    cAMP binds to the catabolite activator protein (CAP), and bound CAP promotes a higher rate of lac operon transcription. So if glucose levels are high, cAMP levels are low, and CAP doesn't activate, so transcription rate of the operon is low, and glucose is metabolised instead.

  1. Chromosomes and DNA

    haemophilia Sickle-cell anaemia Sickle-cell anaemia is caused by a recessive allele for the haemoglobin gene. This results in the production of abnormal haemoglobin. Individuals homozygous for this allele suffer sickle-cell anaemia. Red cells that contain the abnormal haemoglobin change shape and become sickled when the oxygen levels are low.

  2. GENE EXPRESSION

    The mechanism is not known but the duration for heat shock is critical and has been opitmized for the type of bacteria used and the transformation conditions employed. Recovery - this period follows the addition of LB nutrient broth allows the cells to grow and express the ampicillin resistance protein

  1. Investigating the colour variation of Littorina littoralis and their abundance across the upper, middle ...

    This may be the case as the environment becomes drier, the Littorina littoralis will themselves dry out and die, known as desiccation. It was also predicted that towards the lower zone, the darker coloured shells would be more abundant. This may be because the environment is predominantly dark coloured, with

  2. Gm foods and Gene therapy

    it has really big importance, which has to be taken with all seriousness. A recognisable paradigm shift is taking place. No longer is biotechnology able to depend upon a public agreement of the Enlightenment model of development rode by a specific conquering of the nature power.

  1. Is there a Gay Gene?

    This made the researchers investigate the X chromosome. Hamer recruited 40 pairs of homosexual brothers and took DNA samples from each of them preforming genetic linkage analysis using markers. 33 pairs out of the 40 shared a set of five markers located near the end of the X chromosome in an area known as Xq28.

  2. The human Genome Project was designed to identify all the approximate 20,000-25,000 genes in ...

    Also it would help to reduce the likelihood of heritable mutations. * Evolution, and human migration- Human genome project will help us to study migration of different population groups based on female genetic inheritance along with mutations on the Y chromosome to trace lineage and migration of males and to

  • Over 160,000 pieces
    of student written work
  • Annotated by
    experienced teachers
  • Ideas and feedback to
    improve your own work