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