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cDNA

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by

Zhaoyue Xu

on 15 March 2013

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Transcript of cDNA

Creating the Library Once mRNA is purified via one of several different methods (eg. Trizol extraction, column purification), a complementary primer binds to the poly-A tail providing a free 3'-OH end that can be extended by reverse transcriptase to create the complementary DNA strand. mRNA is then removed by using a RNAse enzyme leaving a single stranded cDNA. This cDNA is converted into a double stranded DNA using DNA polymerase using an open 3'-OH end. This comes from the cDNA itself. The polymerase extends the 3'-OH end and later the loop at 3' end is opened. Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids. The cloned bacteria are then selected. Bacteria stocks are then created, grown and sequenced to compile up the cDNA library. Step Four Step Five The mixture of mature mRNA stands are extracted from the cell Step Three The pre-mRNA gets it's introns removed and a Poly-A tail and 5' Methyl-Guanine cap Step Two An eukaryotic cell transcribes DNA into RNA (pre-mRNA) Step One 9 Steps How it's Made Kyle, Xiao, Kevin x2 cDNA A poly-T oligonucleotide primer is hybridized onto the poly-A tail of the mature mRNA template Reverse transcriptase and deoxynucleotide triphosphates is added to synthesize one complementary strand of DNA hybridized to the original mRNA strand. When scientists want to transfer a gene from one cell to another, cDNA is used instead of DNA.

The whole DNA might interrupt the coding sequence for the protein or code for DNA not in the gene (introns). Retroviruses and Microarrays cDNA is often used in gene cloning or as gene probes. Helpful for cloning genes into prokaryotes
that are not usually expressed DNA copies of mRNA usually used to clone eukaryotic genes into prokaryotes. cDNA (complementary
DNA) Compiling cDNA Libraries What does it do? Purpose How does it happen? Step Six To synthesize an additional DNA strand, a RNA of the hybrid strand is digested Step Seven After RNA is digested, a single stranded DNA (ssDNA) is left. Since single stranded nucleic acids are hydrophobic, it loops around itself. Therefore ssDNA froms a hairpin loop at the 3' end. Step Eight The DNA polymerase can then use the hairpin loop as a primer to transcribe a complementary sequence for the ss cDNA Step Nine A double stranded cDNA with identical squence as the mRNA of interest is left. cDNA is also made by retroviruses such as HIV-1, HIV-2, and Simian Immunodeficiency Virus. A retrovirus is an RNA virus that replicates in a host cell. These viruses integrate the reverse transcribed cDNA (from their RNA) into the hosts genome and creates proviruses.

A cDNA microarray (or biochip) is a collection of microscopic spots of DNA attached to a solid surface (such as a slide). This can then used to be hybridize cDNA samples under condotions of high stringency. DNA Biochip Sources http://www.mun.ca/biology/desmid/brian/BIOL4900/CB18_31.html

http://upload.wikimedia.org/wikipedia/commons/0/08/Biochip.jpg

Biology by Raven and Johnson 8th Edition

http://www.accessexcellence.org/RC/VL/GG/retrovirus.php
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