Genomic library construction

Difference between Genomic and cDNA librart. step wise construction of gene library

1. Construction of DNA Library Gurvinder kaur P hD

2. What is DNA library? Collection of  DNA  fragments that have been cloned into vectors so that researchers can identify and isolate the DNA  fragments that interest them for further study. In   molecular biology , a  library  is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. 

3. What are libraries for? Storage/ store many copies of a gene Host most commonly e.coli are use for storing whole genomic data in the form of bacterial colonies Collection of all the clonned vectors Eg genomic library in a petri dish.

4. Why we require libraries? In order to study a gene, a researcher needs to isolate it from all the other genes in an organism's  DNA To make the research easier Once, we have a library, we can locate it by various screening methods and can use it for various research purposes Genomic libraries  are commonly used for sequencing  applications . Ease of purification, storage and analysis.

5. DNA library

6. Genomic Library Contains DNA fragments representing entire genome of an organism Created using molecular cloning vectors are engineered to carry the DNA fragments. vector genome containing the foreign insert is replicated, producing clones of the original genome This collection of clones, in theory contains all sequences found in the original source, including the sequence of interest Genomic libraries can be constructed using various hosts like plasmids, bacteriophage lambda and many more.

7. Steps of genomic library construction Isolation of DNA from cells Digestion into small fragments Introduction into suitable vectors Insertion into bacteria Production/identification of clones Collection of Genomic DNA library

8. 1. Isolation of DNA

9. 2.Digestion into small fragments Purified DNA consist of extremely long strands To begin, the strands must first be cut into manageable sizes Physical shearing: pipetting ,, mixing Restriction enzyme digestion- partial digestion is preferred to get a greater lengths of DNA fragments. The restriction enzyme cut the DNA into 1000s of smaller fragments, each of which may contain one or more gene. Selection of restriction enzymes is very critical

10. 3.Introduction into suitable vectors Each fragment is different and have a unique DNA sequence inserted into suitable vectors including plasmids and bacteriophage vectors Vectors are digested with the same Restriction enzymes and sealed to human DNA using DNA ligase enzyme. The resulting molecules are recombinant.

11. 4.Insertion into host Inserted into host bacteria ( E.coli ) Bacterial cells are made competent to take up the DNA. They replicate their genome along with the vector genome contained with them. Produce clones of the original genome This collection of clones which contains all the sequences, including the sequence of interest forms the genomic library.

12. Multiplication and production of clones

13. Genomic DNA Library-overview

14. Contig

15. cDNA Library Libraries that represent the mrna in a particular cell or tissue are termed cdna libraries. DNA copies derived from the mrna molecules This process is accomplished using enzyme reverse transcriptase, which is isolated from RNA- containg retroviruses. Cdna is synthesized in two steps from mrna molecule. The resulting cdna molecules are then engineered so that they have RE sites at each end of every molecule, which allows them to be digested and inserted into a vector.

16. Steps to prepare cdna

17. Synthesis of cDNA from mRNA

18. Construction of cdna library

19. Differences between a genomic and cDNA library cDNA Library Expressed genes Transcription start sites Open reading frames (ORFs) Splice points Genomic Library Promoters Introns Intergenic Non-expressed genes

20. Constructing a genomic library in  phage

21. Constructing a genomic library in cosmids

22. Constructing a genomic library in YACs

23. Screening a clones Expression screening or Direct selection of recombinants Insertional selection inactivation method Blue white screening Colony hybridization PCR screening of gene libraries Hybrid select/arrest translation Screening expression cDNA libraries.

24. Expression screening

25. Colony hybridization

26. Hybrid arrest/release Individual Cdna clones or pools of clones can be used to hybridize to mrna preparation Hybrid arrest : translate the Mrna population directly, and the inhibition of translation of some products detected. Hybrid release translation : purify the hybrids and the hybridized mrnaa released from them and translated, it identifies the protein encoded by the cdna clone.

27. Uses of gene library