Here is your short paragraph on Genomic DNA clones.
The genomic DNA clones containing fragments of chromosomal DNA with promoter gene are developed to study the regulation of transcription of gene.
Genomic DNA clones are much larger than cDNA clones as they contain both Exon and Intron sequences. The cloning vectors for isolation of genomic sequences can accomodate large piece of DNA.
Generally vectors based on the bacteriophage lambda (λ) are used as they accept large DNA fragments of about 20,000 base pairs. Large DNA fragments are developed by digesting genomic DNA with a restriction enzyme for a short time.
The large fragments are produced by partial digestion. The genomic DNA fragments are joined to bacteriophage vectors in the normal way. Bacteria are infected by bacteriophage with low efficiency of infection so that each bacterium is infected by one phage particle. The collection of bacteria produced in this way is called a genomic DNA library.
To select the genomic DNA sequence of interest, the library is plated onto a layer of cultured bacteria so that many copies of recombinant phages are produced. The recombinant phage multiply inside host bacterial cell. The cell lyses and bacteriophages are spread to surrounded layer of bacteria and infect them.
The dead cells give rise to a clear area on the culture plate called plaque. Each plaque contains many copies of a recombinant bacteriophage that can be transferred to a membrane. Specific DNA clones are selected by incubating the membrane with a radiolabeled cDNA probe complementary to the genomic sequence.
This produces a radioactive area on the membrane which is identified by autoradiographic technique. If a protein product of a particular gene is unknown, it is impossible to isolate an appropriate cDNA clone using conventional techniques. Reverse genetics is applied with a genome and a protein product is identified. It is very time consuming and difficult process.
Direct gene transfer, electro portion and Microprojectile gun:
For monocot plants the Ti plasmid cannot be used. There direct gene transfer is attempted; involving the addition of selectable marker genes together with polyethylene glocyl which stimulates membrane fusion.
Here a short pulse of high intensity electrical current is given which disrupts the all membrane rendering permeability to DNA molecule. The process is successfully employed in 1990 in maize plants.
This is a device used to shoot DNA coated microprojectiles (tungsten particles of 1-4 pm diam) into plant cells.