cdna libraries

7/28/2019 cDNA Libraries

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cDNA Libraries

PHSC 326

Spring 2003


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Preview

Definition of cDNA

Uses of cDNA libraries

Making cDNA

Making cDNA libraries


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Definition

cDNA is complementary DNA

Complementary to mRNA

Copy of expressed protein gene

Made with reverse transcription


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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 DNAViral polymerase made by host


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Reverse Transcriptase

Reverse transcriptase

RNA-dependent DNA polymerase

Require template, primer, Mg++

and dNTPViral 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)


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Making cDNA

Supply RNA template

Supply primer

Supply reverse transcriptase Supply other reagents

(makes single-stranded DNA)


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Uses of cDNA

Obtaining coding sequences

Eukaryotic genes are interrupted

Intron sequences must be removedSpliced exons are mature mRNA

Splicing

Translation


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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


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cDNA vs. Genomic

cDNA

Only protein coding

Expressed proteins

Source specific

Organ

Tissue

Cells

Engineered

Genomic

All DNA of organism

Expressed and silent

Genes and regulatory

Any source ok

Natural


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Making cDNA (in lab)

Purify template

Make DNA strand complementary to

mRNA Destroy template

Make second DNA strand complementary

to first strand


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Purifying mRNA

Poly-adenylated

mRNA

Remove from other cell

components

5

AAAAAAA3

TTTTTTT3bead

Affinity chromatography

Resin with oligo dT

Selective binding

Selective elution


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Affinity Purification

5

AAAAAAA3

5

AAAAAAA3

5

AAAAAAA3

3TTTTTTT

3TTTTTTT

3TTTTTTT


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Making 1st Strand cDNA

Polymerase reaction

Copy template

From mRNA to DNA

Need:

Polymerase

Primer

Nucleotides

Buffer (salts, ions, etc.)

5

AAAAAAA3

3TTTTT5


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Polymerase reaction

Copy template

From mRNA to DNA

mRNA

DNA

5

AAAAAAA3

TTTTT5

3


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Polymerase reaction

Copy template

From mRNA to DNA

Need:

Polymerase

Random primers

Nucleotides

Buffer (salts, ions, etc.)

5

AAAAAAA3

3ATCAGCT5

3ATCTAC5

3TTTACT5

5

3


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Polymerase reaction

Copy template

From mRNA to DNA

5

AAAAAAA3

3ATCAGCT5

3ATCTAC5

3TTTACT5

5

3


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Destroy Templates

Destroy mRNA

RNase H

For cloning, must be

double-stranded DNA

Replace mRNA with

DNA5

AAAAAAA3

5

AAAAAAA3

5

AAAAAAA3


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Replace Templates

RNA fragments act as

primers DNA polymerase I

Copies 1st strand

Nick mRNA

RNase H

Low [RNase H]

5

5

53

3

35

3

5

5

3

3


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Make 2nd cDNA Strand

DNA in 2nd strand is

fragmented Polymerase cant join

Extend primers

DNA polymerase adds onto RNA fragments

RNase H continues to

degrade RNA All RNA replaced with

DNA

5

5

53

3

35

3

5

5

3

3


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Finishing 2nd cDNA Strand

2ndstrand is short at 5end

No primer

Sequence lost

Polish 3 overhang tomake blunt end

Ligase joins fragments

E. coli ligase

5

5

53

3

35

3

5

5

3

3


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Making Libraries

Ligate cDNA fragments into vector

Plasmid vector or viral

Avoiding blunt-ended ligationInefficient

Some inserts will be lost (bad)

Create sticky ends on cDNA


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Linkers

Short dsDNA

Usually 10-15 base pairs

Contains a restriction site

Ligate to cDNA

Large molar excess of linkers

All cDNA molecules ligated

5ATCGAATTCTA3

3TAGCTTAAGAT5


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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 cDNABefore addition of linkers- only linkers cut


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Adaptors

Short semi-dsDNA

Precut sticky ends

Choose with compatible ends for vector

Ligate blunt ends to cDNA

Other end sticky for vector

Problem: sticky ends are self-compatible

5ATACGATG3

3TATGCTACTTAA5


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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 kinaseOther end now can ligate to vector


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Review

cDNA libraries contain cDNA clones

Only protein coding genes

Copy of mRNA only- no silent DNACreate cDNA with several enzymes

Good for isolating protein coding sequences

Express proteins for therapiesStudy function of proteins

cdna libraries

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