cDNA Libraries
PHSC 326 Spring 2003
Preview
Definition of cDNA
Uses of cDNA libraries Making cDNA
Making cDNA libraries
Definition
cDNA is complementary DNA
Complementary to mRNA Copy of expressed protein gene Made with reverse transcription
Reverse Transcription
Not found in normal living cells
All use DNA as genetic material Transcribe DNA into RNA
Some viruses use RNA as genetic material
Retroviruses Viral RNA transcribed into DNA Viral polymerase made by host
Reverse Transcriptase
Reverse transcriptase
RNA-dependent DNA polymerase Require template, primer, Mg++ and dNTP Viral RNA transcribed into DNA Host processes DNA like normal virus
(transcribe DNA into RNA) (make coat proteins by translating RNA) (package RNA into coat and release)
Making cDNA
Supply RNA template Supply primer Supply reverse transcriptase Supply other reagents (makes single-stranded DNA)
Uses of cDNA
Obtaining coding sequences
Eukaryotic genes are interrupted Intron sequences must be removed Spliced exons are mature mRNA
Splicing
Translation
Uses of cDNA
Making protein products
Put cDNA clone into cell Cell transcribes and translates
Understanding protein function
cDNA explains expression sequence Sequence determines function
cDNA vs. Genomic
cDNA
Only protein coding Expressed proteins Source specific
Organ Tissue Cells
Genomic
All DNA of organism Expressed and silent Genes and regulatory Any source ok Natural
Engineered
Making cDNA (in lab)
Purify template Make DNA strand complementary to mRNA Destroy template Make second DNA strand complementary to first strand
Purifying mRNA
Remove from other cell AAAAAAA3 components
5
Poly-adenylated mRNA
Affinity chromatography Resin with oligo dT
bead TTTTTTT3
Selective binding Selective elution
Affinity Purification
AAAAAAA3 3TTTTTTT 5
AAAAAAA3 3TTTTTTT
5 AAAAAAA3 3TTTTTTT 5
Making 1st Strand cDNA
Polymerase reaction
Copy template From mRNA to DNA AAAAAAA3 3TTTTT5 5
Need:
Polymerase Primer Nucleotides Buffer (salts, ions, etc.)
Making 1st Strand cDNA
Polymerase reaction
Copy template From mRNA to DNA 5 3 mRNA DNA AAAAAAA3 TTTTT5
Making 1st Strand cDNA
Polymerase reaction
Copy template From mRNA to DNA AAAAAAA3
5
Need:
Polymerase Random primers Nucleotides Buffer (salts, ions, etc.)
3ATCAGCT5 3ATCTAC5
3TTTACT5
Making 1st Strand cDNA
Polymerase reaction
Copy template From mRNA to DNA 53 AAAAAAA3
5
3ATCAGCT5 3ATCTAC5
3TTTACT5
Destroy Templates
For cloning, must be double-stranded DNA AAAAAAA3 Replace mRNA with DNA
AAAAAAA3
5
Destroy mRNA
AAAAAAA3
RNase H
Replace Templates
Nick mRNA
53 5 3 5 3 3 5 3 5 3 5 RNase H Low [RNase H]
RNA fragments act as primers
DNA polymerase I Copies 1st strand
Make 2nd cDNA Strand
Extend primers 3 5 3 5 3 5
DNA polymerase adds on to RNA fragments RNase H continues to degrade RNA All RNA replaced with DNA
53 5 3 5 3
DNA in 2nd strand is fragmented
Polymerase cant join
Finishing 2nd cDNA Strand
Ligase joins fragments
E. coli ligase 53 5 3 5 3 3 5 3 5 3 5
2nd strand is short at 5 end
No primer Sequence lost
Polish 3 overhang to make blunt end
Making Libraries
Ligate cDNA fragments into vector
Plasmid vector or viral
Avoiding blunt-ended ligation
Inefficient Some inserts will be lost (bad) Create sticky ends on cDNA
Linkers
Short dsDNA
Usually 10-15 base pairs Contains a restriction site
5ATCGAATTCTA3 3TAGCTTAAGAT5
Ligate to cDNA
Large molar excess of linkers All cDNA molecules ligated
Linkers
Digest with appropriate enzyme
Creates sticky ends Choose linker compatible with vector site
Problem: internal restriction sites
Cut cDNA into pieces (bad)
Solution: methylase treat cDNA
Before addition of linkers- only linkers cut
Adaptors
Short semi-dsDNA
Precut sticky ends Choose with compatible ends for vector
5ATACGATG3 3TATGCTACTTAA5
Ligate blunt ends to cDNA
Other end sticky for vector Problem: sticky ends are self-compatible
Avoiding Self-ligation
Adaptors could allow ligation of cDNA
To each other To itself
Make adaptor with PO4 only at one end
Only allow blunt-ended ligation After ligation to cDNA, use kinase Other end now can ligate to vector
Review
cDNA libraries contain cDNA clones
Only protein coding genes Copy of mRNA only- no silent DNA Create cDNA with several enzymes
Good for isolating protein coding sequences
Express proteins for therapies Study function of proteins
