COMPLEMENTARY DNA (cDNA)

DNA synthesized from messenger RNA (mRNA) in a reaction catalyzed by the enzymes 1. Reverse transcriptase 2. DNA polymerase. Is known as Complementary DNA

> Between A and T---- 2 hydrogen bonds > Between C and G---- 3 hydrogen bonds > Above DNA sequence and its complementary strand. strand.

INTRODUCTION

o cDNA---used--prokaryotes.

cloning

eukaryotic

genes

in

o Scientists transfer the cDNA that codes for the protein to the recipient cell---- for expression o cDNA----also produced by retroviruses (such as HIV1, HIV-2, Simian Immunodeficiency Virus, etc.)

OVERVIEW OF cDNA
o central dogma---- in synthesizing proteins o DNA-----transcribed into mRNA o mRNA------translated into protein. o Difference b/w eukaryotic and prokaryotic genes o eukaryotic genes can contain introns o Introns----not coding sequences o In contrast----exons---are coding sequences) o Introns---must be removed from the RNA primary transcript before it becomes mRNA and can be translated into protein

OVERVIEW CONTINUED. CONTINUED.

o Prokaryotic genes---no introns----RNA is not subject to splicing. o Eukaryotic genes---desired to express in prokaryotic cells. o simplified method---add eukaryotic DNA to a vector(prokaryotic host) o Vector---transcribes DNA---- mRNA---- translated to protein.

OVERVIEW CONTINUED. CONTINUED.

o eukaryotic DNA------ contain introns, prokaryotes lack the machinery to splice them o splicing of eukaryotic DNA---- done prior to adding the eukaryotic DNA into the host o DNA----- made as a complementary copy of RNA, without introns------- is complementary DNA (cDNA).

Intron and Exon in Eukaryotic Cells

exon
promotor

exon intron intron

exon

DNA
5
stop codon

3
Transcription

start codon

5
Processing

mRNA

cap
Splicing poly A tail

Intron deleted
Take place in nucleus

mature mRNA

To cytoplasm
Juang RH (2004) BCbasics

SYNTHESIS of cDNA
o cDNA----- often synthesized from mature (fully spliced) mRNA------ using Reverse transcriptase. o Reverse transcriptase; o Operates on single strand of mRNA o Generates its complementary DNA o RNA bases (A, U, G and C) pair to DNA complements (T, A, C and G respectively).

SYNTHESIS Of cDNA CONTINUED.

 To obtain eukaryotic cDNA whose introns have been removed: o Eukaryotic cell--- transcribes the DNA into pre-mRNA o pre-mRNA strands processed;
o o o Introns----removed poly-A tail----added 5 Methyl-Guanine cap--- added

o Mature mRNA strands--- extracted from cell.

SYNTHESIS Of cDNA CONTINUED.

o poly-T oligonucleotide primer o Hybridized onto the poly-A tail of the mature mRNA template o or random hexamer primers--- added--- which contain every possible 6 base single strand of DNA o And can hybridize anywhere on the RNA o Added--Reverse transcriptase and deoxynucleoside triphosphates (A, T, G, C) o This synthesizes one complementary strand of DNA--hybridized to the original mRNA strand.

SYNTHESIS Of cDNA CONTINUED.

o For additional DNA strand---- digest the RNA of the hybrid strand, using---- enzyme (RNase H) o After digestion of RNA-----ssDNA is left o single stranded nucleic acids---- hydrophobic----tends to loop around itself o ssDNA forms a hairpin loop at the 3' end. o From hairpin loop---- DNA polymerase----then use it as a primer to transcribe---complementary sequence for ss cDNA. o Result---- ds cDNA with identical sequence as mRNA of interest

SYNTHESIS Of cDNA CONTINUED.

o Reverse transcriptase scans------ mature mRNA and synthesizes a sequence of DNA-----that complements mRNA template o This strand of DNA is complementary DNA.

APPLICATIONS OF cDNA
o cDNA---- used o In gene cloning o as gene probes o in the creation of a cDNA library o Scientists---- transfer a gene from one cell into another cell---to express----as a protein in recipient cell, o cDNA---- added to the recipient (instead of entire gene) because o DNA for an entire gene may include DNA that does not code for the protein o or interrupts the coding sequence of the protein (e.g., introns)

VIRUSES
o Some viruses also use cDNA o cDNA used to turn viral RNA into mRNA cDNA mRNA.

o Viral RNA

o mRNA----used to make viral proteins----- virus takes over the host cell.

cDNA Synthesis for RT-PCR Protocol: Reagents

o 5X First Strand Buffer---- Invitrogen, Cat. Y00146 o 10mM dNTP Set----Amersham Biosciences, Cat. 272035-01 o 0.1M DTT----Invitrogen, Cat. Y00147 o Random Primers---Invitrogen, Cat. 48190-011 o RNaseOUT Ribonuclease Inhibitor---Invitrogen, Cat. 10777-019 o SuperScript II RNase H- Reverse Transcriptase--Invitrogen, Cat. 18064-014

PROCEDURE
o Combine 3-5 g total RNA and molecular grade water to 8 l final volume. o Add 3 l Random Primers. o Add 1 l dNTP mix. o V ortex and then spin down tube. o Incubate at 65C for 5 min. o Place tube on ice. o Add 4 l 5X Buffer, 2 l DTT and 1 l RNAseOut.

PROCEDURE CONTINUED....

o Vortex and then spin down tube. o Incubate at 42C for 1 min. o Add 1 l SuperScript II RNase H- Reverse Transcriptase. o Incubate at 42C for 60 min. o Incubate at 70C for 15 min. o Add 180 l molecular grade water. o Use Nanodrop 1000 to measure concentration. Set sample type setting to Other Sample and the constant to 33. o Store at -80C

NOTES

 10 mM dNTP Mix: o 10 l dATP [100mM] o 10 l dCTP [100mM] o 10 l dGTP [100mM] o 10 l dTTP [100mM] o 60 l molecular grade water

GLOSSARY
o Retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome o Reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA o Transcription is the process of creating a complementary RNA copy of a sequence of DNA

o Messenger RNA (mRNA) is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes.

GLOSSARY CONTINUED

o Translation---messenger RNA (mRNA) produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein o Intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA o Exon is a nucleic acid sequence that is represented in the mature form of RNA molecule after introns have been removed by splicing

GLOSSARY CONTINUED

Splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined

GLOSSARY CONTINUED

o cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism

Two Libraries
Genes in expression mRNA Reverse transcription

cDNA Library vs Genomic Library

Total Gene Chromosomal DNA

Restriction digestion

cDNA

Complete gene

Gene fragments

Smaller Library

Larger Library

Vector: Plasmid

Vector: Plasmid or phage

Juang RH (2004) BCbasics