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Chapter review…. Gene technology. Ethics. DNA recombinant technology. Gene library. Application. Genetic engineering. Gene cloning. Genomic library. cDNA library. Transgenic organism. DNA finger printing. tools. Genetically modified organism. Target gene. Cloning vector.
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Chapter review… Gene technology Ethics DNA recombinant technology Gene library Application Genetic engineering Gene cloning Genomic library cDNA library Transgenic organism DNA finger printing tools Genetically modified organism Target gene Cloning vector Restriction enzyme Modifying enzyme Host cell Polymerase chain reaction methods
At the end of the lesson, you should be able to : • Describe gene library • List the types of gene library
Genomic library… Concept ofgenome “The complete set of DNA complement of an organism’s genes. Every species of organisms have its own genome that difference with another species”
Genomic library… genomic “The study of whole sets of genes and their interactions”
Genomic library… Genomic library A set of thousands of DNA segments from a genome, each carried by plasmid, phage or other cloning vector
Genomic library… • Thousand of different recombinant plasmids are actually produced and a clone of each ends up in a colony. The complete set of thousands recombinant plasmid clones; • Each carrying copies of a particular segments from the initial genome, is refer to as a genomic library
Genomic library… • Genomic library can be enormous. The 3 billion pairs of the human genome. • for example, can occupy millions of plasmids, each harboring a small piece of DNA from one of our chromosomes, with many overlaps.
Genomic library… • A genomic library also contains too much information for a researcher seeking a particular protein-encording gene, it also contains introns, many repeated sequences and the genes that encode rRNAs and tRNAs.
Genomic library… • Gene library included a collection of cloned DNA fragments reprising the entire genetic material of an organism. • This facilitates screening and isolation of many particular gene. Gene libraries are thus repositories of raw materials for use in genetic engineering. • A human gene library may contain some 500 000 separate clones.
cDNA library… Complimentary DNA (cDNA) A DNA molecule made in vitro using mRNA as a template and the enzyme reverse transcriptase.
cDNA library… • A cDNA molecule therefore corresponds to a gene, but lacks the introns present in the DNA of the genome.
cDNA library… Complimentary DNA (cDNA) library A limited gene library using complimentary DNA.
cDNA library… • The library includes only the genes that were transcribed in the cells examined Such a cDNA library represent only part of a cells genome. • This is an advantage if a researcher wants to study the genes responsible for specialized functions of a particular kind or cell.
cDNA library… • Also, by making cDNA from cells of the same type at different times in the life of an organism, one can trace changes in patterns of gene expression. • cDNA represents only protein encording genes. A specific cDNA can be taken from cDNA library and used to isolated the original gene of interest from the genomic library.
Insulin production using cDNA… • The production of human insulin by engineered bacteria (E. coli ) was an important early example for Biotechnology.
Insulin production using cDNA… • Once upon a time, diabetes were treated with regular injections of insulin from animals, like cattle and pig. • But human insulin from animal sources are different chemically (different in only 2 of 51 amino acids), and repeated injections from animal insulin led to allergic reactions in many patients.
Insulin production using cDNA… • Today, human insulin produced by E. coli overcame this problem for many patients and actually save the time and cost of the treatment. • The technique used was through recombinant DNA technology, using E. coli, and the name of this product is Humulin S.
Insulin production using cDNA… How? Step 1 mRNA codes insulin to be extracted from the cells of the pancreas that produce insulin
Insulin production using cDNA… How? Step 2 mRNA processes to removes introns, finally produce a functional mRNA
Insulin production using cDNA… How? Step 3 Reverse transcriptase enzyme is added, this synthesis a cDNA by using mRNA as template. mRNA strand then discarded/ realized by using mRNA-degrading enzyme
Insulin production using cDNA… How? Step 4 DNA polymerase enzymes is added, this synthesizes a second DNA strand, complementary to cDNA in vitro. This step produced the required gene (coding insulin) in cDNA double-stranded
Insulin production using cDNA… How? Step 5 The DNA of gene without introns then isolated from it source and cut with restriction enzyme ( Bam H1 )
Insulin production using cDNA… How? Step 6 A plasmid removed from a bacterial cell is opened up at a specific point using the same restriction enzyme ( Bam H1 ).
Insulin production using cDNA… How? Step 7 The DNA of gene without introns then isolated from it source and cut with restriction enzyme ( Bam H1 )
Insulin production using cDNA… How? Step 8 The DNA is now attached to the plasmid using DNA ligase. The resultant plasmid, carrying the human DNA for insulin. This is called plasmid vector/ recombinant plasmid/ rDNA.
Insulin production using cDNA… How? Step 9 The rDNA then reinserted into the host cell (E. coli). Under the right conditions E. coli takes up the rDNA by transformation.
Insulin production using cDNA… How? Step 10 The actual gene cloning, the production of multiple copies of genes. The bacterium E. coli with its recombinant plasmids, is allowed to reproduce.
Insulin production using cDNA… How? Step 10 Finally, a lot of bacterial clone carrying many copies of the gene for insulin will be produced. Later, the bacterial can be used to produce much insulin for health industry purposes.
