Cytic Fibrosis Research

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The purpose of this experiment was to identify the faulty gene that causes cystic fibrosis through use of cDNA libraries and subsequent other techniques. The cystic fibrosis gene was found to be on chromosome 7, and was approximately 6.5kbp long and found in patients afflicted with cystic fibrosis. The protein thought to be the cause consists of two smaller motifs associated with the membrane and a domain believed to be involved in ATP binding. However a deletion of three base pairs results in the omission of a phenylalanine residue at the centre of the first predicted nucleotide binding domain. Some of the techniques used in the experiment include: cDNA library screening, Western Blots and PCR were used. The purpose of this essay is to discuss all techniques involved, this involves both the description of techniques used and the reasons they were used, also if any newer or superior techniques could be used. The process started with a large, continuous segment of DNA spanning 4 transcribed sequences which was believed to be the cystic fibrosis locus. To determine the separate sequences DNA hybridisation was used and RNA Hybridization subsequently characterised the results. DNA hybridization is the process of combining two complementary single stranded DNA or RNA molecules; they anneal using normal base pairing reactions. This can be used to detect certain sequences by using probes. The probes contain either radioactive of illuminesent materials thus it can be detected later. The probes anneal to the complementary single stranded DNA and RNA and can be visualised later by autoradiography or chemiluminescence. This allows the detection of certain gene sequences and allowing the Cystic Fibrosis gene to be pinpointed and accurately e... ... middle of paper ... ...are then designed to specifically down regulate the gene of interest. Another major technique becoming common use is the Chain terminater or Sanger Dideoxy technique. This depends on the use of dideoxy nucleotides which are analogues of dNTPs. These can be incorporated into DNA strands and can be labelled with a colour for later detection. The lack of 3’ OH group means that after the ddNTP has bonded that no further nucleotides can form phosphodiester bonds terminating polymerisation. The ddNTPs bond randomly thus giving millions of fragments of varying length. These are run on a gel and the labelled ddNTPs are detected. Due to the random nature of the bonding a the full sequence can be recorded. These are the main two new forms of detection but more methods are joining the limelight. Techniques like Illumina, Roche 454 and Ion Torrents are all becoming common use.

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