genomic and cDNA library in recombinanat DNA technology

genomic and cDNA library in recombinanat DNA technology

1. BSc Biotechnology 3 nd year V Semester BBT 502- Recombinant DNA technology Ganesh Nawkar Lecture8: Construction of genomic and cDNA libraries 01/09/2023

2. What is the genomic library? A genomic library is a fragmented DNA collection of the whole genome of an organism. The fragments are stored in bacterial or yeast vectors later on. A clone/collection of similar types of genomic DNA fragments prepared by techniques like restriction digestion is referred to as a genomic library.

3. Properties of a genomic library It should have all the genomic content of the genome of an organism. The size of fragments should be clone-able. The library represents the entire genome. The library should be screenable . Every fragment from the library should be processable enough.

4. Genomic DNA library preparation

5. Genomic DNA library preparation Genomic DNA isolation With the help of standard protocol or using DNA extraction kits The isolation process includes steps of cell lysis, removal of protein and other impurities, DNA precipitation, DNA purification and DNA elution The isolated genomic DNA should have purity around ~1.80 (at 260/280 absorbance) and quantity around or more than 100microgram

6. Genomic DNA library preparation DNA fragmentation DNA extraction is followed by DNA fragmentation, where genomic DNA is parted into smaller random-sized fragments. DNA fragmentation by following methods: Mechanical DNA is fragmented mechanically to produce blunt ends Mechanical processing needs sub-processing such as adaptor ligation and sticky end generation Restriction enzyme Restriction enzymes used to cleave DNA to generate sticky end

7. Genomic DNA library preparation Cloning into a suitable-sized vector Common vectors used for genomic library preparation are plasmid, cosmid , phage, bacteriophage, lambda phage, BAC or YAC.

8. Genomic DNA library preparation Transformation into host cells E.coli and yeast cells are two common organisms used as a host for genomic DNA transformation Screening of genomic library The probe-hybridization and polymerase chain reaction are used for screening genomic fragment inserts

9. Genomic DNA library preparation

10. Advantages The preparation process is comparatively simple and easy. A huge number of data can be generated. Fragments can be analyzed to assess SNPs and mutations. It is a comparatively speedy process The fragment library can be stored for many years. Disadvantages The size of individual fragments is too large; hence sometimes sub-fragmentation is required for some downstream processing. Also, the larger fragment size is difficult to process and treat. The effect of a particular gene or protein can’t be studied. Genomic DNA library

11. Genomic DNA library preparation N = the number of clones that are required p = the probability that any given gene will be present a = the average size of the DNA fragments inserted into the vector b = the total size of the genome