cDNA

– DNA made from mRNA

Why use cDNA probes instead of RNA probes?

– RNA is labile – 2' OH highly reactive

– RNase enzymes – everywhere

– highly stable

– e.g. survive >30 minutes at 100°C

– RNA work requires: – dedicated equipment for RNA

– diethylpyrocarbonate (DEPC) treated solutions

– RNase inactivator

– specific mRNAs often rare – e.g. housekeeping genes

– may have < 5 copies of mRNA in cell at once

– mRNAs may occur in different forms from same gene

– e.g. different intron splicing

– production of RNA in vitro difficult

– unless T7 or T3 promoter present

– requires cloned gene

– cDNA can be cloned – in plasmid or phage vectors

– maintained in stable form

– lots copies from high copy number vectors

– expression vectors

– allow screening of lacZ fusion proteins

– need cDNA for eukaryote gene expression in E. coli

– no intron splicing

– cDNA can be amplified by PCR

– multiple copies of rare mRNAs

– anchored PCR techniques – e.g. RACE

requirements for cDNA

mRNA

– usually isolated from tissue with lots of desired mRNA

– (if possible) – tissue with lots of desired protein

– RNA isolated from tissue (mostly rRNA & tRNA)

– using lysis buffer with guanidinium isothiocyanate

– denatures proteins – including RNases

– most eukaryotic mRNAs have polyA tail

– can be purified away from other RNA molecules

– using oligo-dT cellulose

– matrix + poly-T oligonucleotides

– binds mRNA in high salt, eluted in low salt

– will not work for prokaryotic or histone mRNAs

– no polyA tail

reverse transcriptase enzyme – usually from retroviruses

– has 3 enzyme activities

1. RNA-dependent DNA polymerase

– makes DNA from RNA template – 5'-3' direction

– requires primer (DNA polymerase)

– retroviruses use tRNA as primer

– no proofreading – high error rate (~¹/₅₀₀)

– hence high HIV mutation rate

2. RNase activity – degrades RNA in RNA/DNA hybrid

– removes RNA template

3. DNA-dependent DNA polymerase

– synthesizes second strand of cDNA

– using first strand as template

– inefficient

– second strand synthesis

– usually done using other DNA polymerase

sources of reverse transcriptase

– avian myoblastosis virus (AMV)

– enzyme has maximum activity at 42°C

– reduces problems from secondary structure

– loops from H-bonding – may stop polymerase

– enzyme has strong RNase activity

– may degrade RNA before cDNA can be made

– Moloney strain of murine leukemia virus (Mo-MLV)

– enzyme has maximum activity at 37°C

– secondary structure may be problem

– low RNase activity – produces longer sequences

– gene cloned in E. coli

– mutated derivatives with no RNase activity

– Stratagene, Life technologies

primer – for 1^st strand synthesis

– ideal primer

– gene-specific primer 3' of gene stop codon

– only produce desired cDNA

– must know sequence

– poly-T primer – hybridizes to all polyA mRNAs

– works for most eukaryote mRNAs – not specific

– does not work for prokaryote or histone mRNAs

– unless add poly-A tail with poly(A) polymerase

– adds ATP to RNA 3' end

– hexanucleotide primer

– mix of different 6-bp oligonucleotides

– hybridize at multiple sites

– multiple origins of DNA synthesis

– works for prokaryote mRNAs

– also may help with secondary structure

– enzyme not blocked at one site

primer – for 2^nd strand synthesis

– self-priming method (Fig. 4.2)

– used to make first successful cDNAs

– requires homology:

– between 3' end of 1^st strand and 2^nd site within sequence

– 3'end primes 2^nd strand

– get single-stranded loop at 5' end of gene

– remove with S1 nuclease

– digests single-stranded DNA

– does not work for most mRNAs

– always lose 5' end of gene

– RNase H method (Fig. 4.1) – common method

– treat RNA/DNA hybrid with RNase

– partial reaction – enough to nick RNA strand

– nicked RNA serves as primers for DNA synthesis

– using E. coli DNA polymerase I

– extends RNA primers

– 5'-3' exonuclease degrades RNA in hybrid

– produces blunt ended double-stranded DNA

– cloned in blunt-cut vector

– homopolymer tailing (Fig. 4.4)

– use terminal transferase enzyme

– add poly-dCTP tail to 1^st strand

– use poly-G primer for second strand

– also used to add “sticky” single-stranded ends for cloning

advanced priming techniques (Fig. 4.5)

– incorporate restriction sites in primers – for cloning

– 1^st strand – polyT + RE site – homopolymer tail

– 2^nd strand – polyG + RE site

– end up with molecule having RE sites at ends

– available in kits – multiple cloning sites in primers

– other schemes available