Essay --

BIOT643 Spring 2014- Quiz2 (50 points) Due in TIN and Assignment folder before midnight Sunday Feb 23rd. Please provide short, but specific and complete answers. Q1) For each of the following components, briefly describe how each is used in the construction of DNA libraries. (5 points each) Reverse Transcription is an essential step in constructing a complementary strand, or cDNA library. DNA is converted into mRNA by transcription. In reverse transcription mRNA is incubated at 70 degrees with a primer to denature the structure. This will allow the primer to anneal to the mRNA. After dNTPs and buffer are added to the reaction. Then, transcription occurs at 37 degrees Celsius, followed by the temperature is increased 70 degrees Celsius to inactivate the enzyme (Life Technologies). A 3’ end polyA tail, is used to pull the mRNA from the cells and starting point for reverse transcription in order to make cDNA from the mRNA. Then, the ribonuclease breaks the mRNA to make a produce only a single stranded cDNA (Ebrahimian, 2014). The 5' cap of mRNA strand is replaced with a synthetic RNA oligonucleotide, called 5’-Oligo –Capping. The 3'end PolyA and the 5' Oligo cap are tags to make sure the ends of the mRNA molecule are present in the cDNA. The cDNA, with now a 5' Oligo cap and 3' end polyA, through DNA polymerase is entered into a vector to make cDNA clones (Ebrahimian, 2014). A way to chop an entire DNA of an organism is with restriction enzymes. A restriction enzyme makes small fragments. Two examples of these enzymes used MMLV and SSIII (Life Technologies). The small fragments are then cloned into a vector (Ebrahimian, 2014). In a genomic library, the vector creates a recombinant. Eventually the cloning of numerous recombinan... ...gical is used to identify the protein. You will not use hybridization if a DNA probes, such as homologous and heterologous gene probe is not available. In addition, hybridization screening will be used for large number of clones, if the cDNA clone is not at full length, and synthesized products in the host that are not antigenically of biologically active (Ebrahimian, 2014). Reference: Cloning and Molecular Analysis of Genes. (n.d.). North Dakota State University . Retrieved February 20, 2014, from http://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/clone5.htm Ebrahimian, S. (Director) (2014) Definition of DNA Library. BIOT 643. Lecture conducted from University of Maryland, University College, Adelphi Life Technologies Corp (2012, December 6. Simplified RT- Reverse Transcription Animation. Retrieved February 21, 2014. http://www.youtube.com/watch?v=0MJIbrS4fbQ