Methods For Preparing cDNA From Low Quantities of Cells
AssigneeYale University
InventorsSHERMAN M. WEISSMAN, XINGHUA PAN
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Abstract
Methods for preparing cDNA libraries from single and low quantities of cells are disclosed. The methods are based on the principles of multi-strand displacement amplification or semi-random primed polymerase chain reaction. The methods typically include a step of reverse transcription and subsequent amplification of cDNA. The methods can be adapted for preparation of cDNA libraries that are representative of mRNA or whole RNA expressed by the cell or cells. The cDNA is suitable for sequencing or microarray analysis.
Claims22
A method of preparing a cDNA library from a single cell or a low number of cells, comprising
- (a) preparing single stranded cDNA from cellular RNA by a reverse transcription (RT) reaction comprising
- (1) denaturing the RNA;
- (2) annealing one or more RT primers to the RNA;
- and
- (3) extending the RT primers to form the cDNA;
- (b) optionally preparing double stranded cDNA by second strand synthesis of the single stranded cDNA;
- (c) circularizing the cDNA;
- (d) amplifying the circularized cDNA by a multiple displacement amplification (MDA) reaction comprising annealing one or more MDA primers to the cDNA and extending the MDA primers with a phi29 DNA polymerase for form the cDNA library.
A method of preparing a cDNA library from a single cell or a low number of cells, comprising
- (a) preparing single stranded cDNA from cellular RNA by a reverse transcription (RT) reaction comprising
- (1) denaturing the RNA;
- (2) annealing one or more RT primers to the RNA;
- and
- (3) extending the RT primers to form the cDNA;
- (b) optionally preparing double stranded cDNA by second strand synthesis of the single stranded cDNA;
- (c) amplifying the cDNA by a semi-random primed polymerase chain reaction procedure comprising
- (1) at least four cycles of denaturing the cDNA, annealing a semi-random primer to the cDNA, and extending the semi-random primer with a DNA polymerase, wherein the semi-random primer is a mixture of primers comprising a 5′ sequence capable of self-annealing into a hairpin or hairpin loop structure, 3′ random primer sequence comprising an N stretch wherein “N” can be any nucleotide, and wherein the semi-random primer comprises the universal primer sequence of a polymerase chain reaction (PCR) primer;
- (2) digesting or removing excess random primer;
- (3) at least ten cycles of PCR comprising denaturing, annealing, and extending the PCR primer to form a cDNA library.
Dependencies are inferred from the claim text.