Lecture-4H

Lecture-4H - #4 Recombinant DNA technology (I) 1. 2. 3. 4....

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#4 Recombinant DNA technology (I) 1. Restriction enzymes 2. DNA cloning and vectors 3. DNA libraries 4. Gene cloning/isolation
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Major developments in recombinant DNA technology 1960’s: Vectors, hybridization methods 1970’s: Restriction enzymes, DNA sequencing 1980’s: Transgenic technology, PCR 1990’s: Genomics and bioinformatics 2000’s: System biology Problems in working with DNA To study a gene, we need to isolate the gene in its pure form. But genes are all connected in vast DNA molecules, each gene is a very small fraction of the entire genome. Unlike protein (each protein is different chemically), every gene is similar chemically. So a gene can not be easily distinguished and isolated by conventional physical or chemical means. Therefore, genes are usually studied in the form of recombinant DNA (a DNA molecule originated from different sources) cloned in various vectors.
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Clone : Collection of cells originated from a single cell. Collection of cells containing the identical type of DNA Collection of identical DNA fragments. ...... Collection of anything that is identical to each other and to the parent (we are not the clone of either of our parents) are called clones. We often want to clone only a piece of a huge genome at a time (e.g. to study one gene). A long DNA fragment can be cleaved into smaller pieces at specific sites by restriction enzymes - Restriction fragments can be connected together by DNA ligase, resulting in recombinant DNA - If a DNA fragment is successfully ligated to a vector and propagated in a host to allow purification of the DNA, it is cloned. Clone and cloning
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Bacteria have a defense system called restriction system. It is composed of two types of enzymes: a DNA methylase and a DNA endonuclease called restriction enzyme. A bacterium methylates its own DNA at specific sites called restriction sites, the methylated DNA cannot be cut by the restriction enzyme. Foreign DNA (such as viruses) are not methylated at the restriction site associated with the host restriction system (although it may be methylated at other sites). The restriction enzyme cleaves the foreign DNA at the restriction site and destroy the foreign invader. 1. Restriction system and restriction enzymes
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Restriction enzymes 1. Restriction enzymes are bacterial endonucleases that usually recognize specific 4 to 8 base pair sequences in double stranded DNA. 2. Restriction enzymes cleave both DNA strands at the restriction sites in a symmetric manner. Restriction enzymes cut the DNA with the specific sequence into a reproducible set of fragments called restriction fragments. 3. Many restriction enzymes each with different recognition sequences are commercially available.
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usually short inverted repeat sequences known as palindromes (palindromes are words that read alike forwards and backwards) Restriction site Why palindrome? Restriction enzymes usually
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Lecture-4H - #4 Recombinant DNA technology (I) 1. 2. 3. 4....

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