Construction of cDNA involves three steps:

1)Synthesis of first strand from mRNA

2)Removal of RNA template

3)Synthesis of second strand from first

strand

Development of cDNA cloning

strategies

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1 An early cDNA cloning strategy

a hairpin loop

the first cDNA strand has the tendency to transiently fold back on itself.

A serious disadvantage of the hairpin method is that cleavage with S1 nuclease results in the loss of a certain amount of sequence at the 5′ end of the clone.

2 Improved method

a full-length cDNA

For cDNA expression libraries, it is advantageous, if the cDNA can be inserted into the vector in the correct orientation.

using an oligo-dT primer containing a linker sequence when the second strand is primed. oligo-dT primer

There are 3 methods to achieve direction cloning for expression

adding a linker to the double-stranded cDNA before the hairpin loop is cleaved

using primers for cDNA synthesis that are already linked to a plasmid cDNA

3 adding a synthetic linker to the double-stranded cDNA before the hairpin loop is cleaved

4 using an oligo-dT primer containing a linker sequence

direction cloning achieved

5 using primers that are already linked to a plasmid

a simple and general method for non-directional cDNA cloning.

nick-translation

The Gubler–Hoffman method---widely used

high efficient

Limitations of conventional clone strategies (of cDNA libraries)

1 trigger a 3′-end bias

Two drawbacks

be addressed through the use of random oligonucleotide primers, for both first- and second-strand cDNA synthesis.

oligo-dT primers are used to initiate first-strand synthesis,

Full-length cDNA cloning

partly due to deficiencies in the reverse-transcriptase enzymes used for first-strand cDNA synthesis.

2 difficult to isolate full-length clones (when the size of a cDNA increases)

Native enzymes have poor processivity and intrinsic RNase activity,

leads to degradation of the RNA template

generate libraries (highly enriched in full-length clones).

Combining cap selection and nuclease treatment

to select for full-length first-strand cDNAs

all eukaryotic mRNAs have a 5′end cap

Strategy

Selection of 5 ′mRNA ends

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The capture method of full-length cDNA cloning,

using the eIF-4E to select mRNAs with caps

an oligo-dT primer

hybrid molecules

protecteddigested away

eukaryotic initiation factor

digests single-stranded RNA

full-length molecules be ligated to a specific oligonucleotide

An oligo-capped population of full-length mRNAs resulted

An alternative method, oligo-capping

broken and degraded molecules cannot be ligated to a specific oligonucleotide

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alkaline phosphatase

acid pyrophosphatase

oligo-capping

removes the cap

The basis of the method is thatRNA is treated with alkaline phosphatase and acid pyrophosphatase.

removes phosphate

an oligo-capped population of full-length mRNAs.

using the oligo-dT primer and a primer annealing to the oligonucleotide cap.

PCR as an alternative to cDNA cloning

RT-PCR

amplify RNA sequences in cDNA form.

Using a specific 3′ primer to generate

the first cDNA strand, initiate the PCR amplification by adding a

5′primer to the reaction mix.used the total RNA as the starting

material.

Features of Reverse transcription

RT-PCR can be used to provide the cDNA for library construction, when the source is unsuitable for conventional approaches.

universal primers

amplification of all mRNAs

subcloned into suitable vectors.

cDNA library

library may contain many mutations

Disadvantages of the PCR-based approaches(3)

certain amount of distortion may produced

the DNA polymerases are more error-prone

Competition among templates, and a bias towards shorter cDNAs.

contaminating genomic sequences.

depend upon specific primers

be amplified and false results resulting

screening a cDNA library is laborious,

may not yield a cDNA clone with a full-length coding region,

the sought-after cDNA may be very rare

disadvantage of building cDNA library