DNA Cloning

Cloning:

1. Cell fractionation by conventional chromatographic methods to yield about a microgram of protein.
2. The protein is analyzed to yield the identity of the first 30 amino acids - its N-terminal amino acid sequence.
3. The Genetic Code is used to predict nucleotide sequences corresponding to this amino acid sequence.
4. DNA fragments complementary to these sequences (oligomers of 15-20 bases) are chemically synthesized.
5. The DNA fragments are then hybridized (base-paired) with total cellular mRNA.
6. Long cDNA segments are produced from mRNAs complementary to the DNA fragments using the enzyme reverse transcriptase.
7. Large amounts of this cDNA is obtained by cloning into plasmids (amplification).
8. Selection of the right clone (several steps).
9. Finally, the cloned cDNA is incorporated into an expression vector or plasmid an_d transferred into bacterial or yeast cells.
10. This is the starting point for scaled-up production of large amounts of the protein.