1. BSL2016 / 2018 – Lecture 7 – cDNA libraries cDNA synthesis results in the generation of 1000’s of cDNA molecules. All these cDNA molecules are derived from individual mRNA’s mRNA’s are the product of active, transcribed, genes ( including, in our example, the product of the gene coding for the cysteine rich protein. cDNA molecules have to cloned in order to characterise them, and to find the cDNA molecule you are after. cDNA molecules are usually (but not always) cloned into plasmids

2. BSL2016 / 2018 – Lecture 7 – cDNA libraries cDNA populations have to be cloned. When they have been inserted into plasmids which are used to transform bacterial cells, you have a population of transformed bacterial cells each of which contains a cDNA molecule. This is called a cDNA Library. An excellent cloning vector is TOPO TA (Invitrogen)

3. BSL2016 / 2018 – Lecture 7 – cDNA libraries 3´ T-overhangs for direct ligation of PCR products EcoR I sites flanking PCR product insertion site. Kanamycin and ampicillin resistance genes for selection in E. coli LacZa fragment for easy blue/white colony screening

4. BSL2016 / 2018 – Lecture 7 – cDNA libraries Blue/White selection (lacz inactivation) allows the easy identification of recombinants.

5. BSL2016 / 2018 – Lecture 7 – cDNA libraries Once a cDNA library has been created, it must be screened for the gene of interest to identify a particular cDNA clone Standard cDNA libraries are screened by colony hybridisation using homologous or heterologous DNA probes. Homologous probes: Probes which are 100% identical to the cDNA you are screening for. Isolate protein, sequence it, deduce DNA sequence from the aa seq. CYS-CYS-ARG-GLU-VAL-THR TGC-TGC-CGC-GAA-GCC-ACA

6. BSL2016 / 2018 – Lecture 7 – cDNA libraries Heterologous DNA probes are not 100% identical to the cDNA you are trying to isolate, but sufficiently similar to form a DNA/DNA hybrid Heterologous probes are usually sequences which have been cloned from one species and which are similar to genes from other species A cysteine rich seed gene from soybean will be similar in sequence to a cysteine rich gene from pea – both are legumes and the proteins in the seeds of both are very similar. So, a cysteine rich gene from soybean will hybridise to its homologue in the pea cDNA library.

7. BSL2016 / 2018 – Lecture 7 – cDNA libraries For probes to hybridise to their targets in a colony hybridisation, the probes have to be labelled (radioactive or non-radioactive) Two main labelling techniques random priming and nick translation:

8. BSL2016 / 2018 – Lecture 7 – cDNA libraries

9. Colony hybridisation is a technique where radioactive probe sequences are used to identify bacterial colonies carrying the gene of interest.

10. BSL2016 / 2018 – Lecture 7 – cDNA libraries An actual colony hybridisation result :

11. BSL2016 / 2018 – Lecture 7 – cDNA libraries If you have already isolated the protein, another way of finding the gene which codes for it is to perform an “Expression screen”. First, you need to raise antibodies against your protein. Antibodies will bind to bacterial colonies expressing “your” protein.