Details regarding synthesis of genomic library and its screening procedures.

1. Construction and Screening of
Genomic library
Dr. Mayank Chaudhary
Assistant Professor
Department of Biotechnology
Maharishi Markandeshwar (Deemed to be University)
Mullana-Ambala, Haryana, INDIA

2. • Genomic library is specific collection of DNA covering entire genome of
an organism. It covers all DNA sequences such as expressed genes, non-
expressed genes, exons and introns, promoter region and intervening DNA
sequences.
• Construction of genomic library involves isolation, purification and
fragmentation of genomic DNA followed by cloning of fragmented DNA in
suitable vector.
• Phenol-chloroform method is used for isolation and purification of
eukaryotic genomic DNA.
• Fragmentation is done by physical or enzymatic method.

3. • Physical method: It involves mechanical shearing of genomic DNA by
syringe needle or sonication to break DNA molecules in size suitable for
cloning.
• Enzymatic method: It involves use of restriction enzymes for fragmentation
of purified DNA. RE can result in either blunt ends or sticky ends. Blunt
ends are converted to sticky ends prior to cloning by use of Linkers and
Adapters.

4. • Vectors used for cloning large DNA fragments: Lambda phage, YAC, BAC.
• Lambda replacement vectors include Lambda EMBL series are widely used
for construction of genomic library.

5. Sub-genomic library: Library which represents only a fraction of the
genome.

6. Applications of genomic library:

7. Screening of genomic library
• Screening is the process of identification of clones carrying gene of
interest. Screening of libraries can be done by following approaches:
1) Detecting a particular DNA sequence.
2) Gene expression.
Methods of screening based on detection of DNA sequence:
1) Screening by hybridization.
2) Screening by PCR.
Screening methods based on gene expression:
1) Immunological screening.
2) Screening by functional complementation.

8. • Screening by Hybridization: It is most commonly used method of library
screening. It relies on hybridization of single stranded probe to targeted
complementary sequence.
• Commonly used methods of hybridization are:
1) Colony hybridization.
2) Plaque hybridization.

9. • Colony hybridization: It is also known as replica plating and is used to
screen plasmid or cosmid based libraries.

11. • Plaque hybridization: It is also known as plaque lift and used for isolation
of recombinant phage by nucleic acid hybridization

12. • Screening by PCR: It is employed for identification of rare DNA sequences
in complex mixtures. It is labor intensive process.
• Immunological screening: It involves the use of antibodies to recognize
antigenic determinants on the polypeptide. As a result, it does not rely on
particular function of expressed protein.
• Secondary antibody recognizes constant region of primary antibody and is
conjugated to enzymes (horseradish peroxidase, alkaline phosphatase).

14. • Screening by functional complementation: It is the process of
compensating a missing function in a mutant cell by particular DNA
sequence for restoring wild-type phenotype. Eg. Functional
complementation in transgenic mice for isolation of shaker-2 gene.

15. REFERENCES
1) T.A. Brown. Gene Cloning and DNA Analysis-An
Introduction. Seventh Edition.
2) NPTEL notes (Module 4).