Gene+Splicing

//__1. Recombinant DNA technology or DNA cloning:__// The process where a DNA fragment is transferred from one organism to a genetic material that self-replicates such as a bacterial plasmid. The DNA fragment then can be placed in a foreign host cell. This technology started around the 1970’s. In order to “clone a gene” the gene in the DNA fragment needs to be isolated from the chromosomal DNA; this is done by using restriction enzymes, then combined with a plasmid with the same restriction enzyme. The “recombinant DNA molecule” refers to the combination of the fragment form the chromosomal DNA that is to be cloned and the vector DNA.

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Gene splicing is cutting the DNA of a gene to add base pairs. Chemicals called restriction enzymes cut the DNA. Thousands of varieties of restriction enzymes exist, each recognizing only a single nucleotide sequence. Once it finds that sequence in a strand of DNA, it attacks it and splits the base pairs apart, leaving single helix strands at the end of two double helixes. Scientists are then free to add any genetic sequences they wish into the broken chain and, afterwards, the chain is repaired with another enzyme called ligase.
 * __What is gene splicing?__**
 * __How does this happen?__**

There are serval types of common alternative splicing events: //Exon Skipping:// This is the most common known alternative splicing mechanism in which exon(s) are included or excluded from the final gene transcript leading to extended or shortened mRNA variants. //Intron Retention:// An event in which an intron is retained in the final transcript. In humans 2-5 % of the genes have been reported to retain introns. //Alternative 3' splice site and 5' splice site:// Alternative gene splicing includes joining of different 5' and 3' splice site. In this splicing mechanism, two or more alternative 5' splice site compete for joining to two or more alternate 3' splice site.
 * __Gene Splicing Mechanism__**

//Gene splicing// leads to the synthesis of alternate proteins that play an important role in the human physiology and disease. Currently, the most efficient methods for large scale detection of splice variants include computational prediction methods and microarray analysis. Microarray based splice variant detection is the most popular method currently in use. The highly parallel and sensitive nature of microarrays make them ideal for monitoring gene expression on a tissue-specific, genome-wide level. Microarray based methods provide a robust, scalable platform for high-throughput discovery of alternative gene splicing. A number of novel transcripts were detected using microarray based methods that were not detected by ESTs using computational methods. Another commonly used method for discovering novel isoforms is RT-PCR followed by sequencing. This is a powerful approach and can be effectively used for analyzing a small number of genes. However, it only provides only a limited view of gene structure, is labor-intensive, and does not easily scale to thousands of genes or hundreds of tissues.
 * __Splice Variant Detection Methods__**

Any form of genetic material can be spliced together; such as bacteria and chicken DNA for example. As in the video Pandora’s Box, splicing is used for the production of insulin and growth hormone to cure human maladies. In the past, insulin was only obtainable from the pancreas of cadavers, requiring 50 cadavers to yield one dose. With modern splicing techniques, enough insulin can be produced for all diabetics. The insulin-producing genes from human DNA are spliced into plasmid DNA; the plasmids are then allowed to infect bacteria, and, as the bacteria multiply, large amounts of harvestable insulin are produced. Splicing has other practical medicinal uses, too. In July of 1996, a 68-year-old woman became the first patient to be treated for arthritis (a disease which affects an estimated 2.1 million Americans) via gene therapy. At the University of Pittsburgh, therapeutic DNA that blocks the production of a specific protein (IL-1) that causes arthritis pain was injected into two of her knuckles.
 * __What implications does this have?__**

Simulation of gene splicing: Make a lab where you can do your own gene splicing! [|Simulation of Gene Splicing]
 * - Rachel Fletcher

http://www.accessexcellence.org/AE/AEPC/WWC/1994/simulation_gene_splicing.html**
 * http://www.premierbiosoft.com/tech_notes/gene-splicing.html**