An endonuclease will digest a nucleic acid molecule anywhere in the middle of the chain. The restriction endonucleases are one of the site-specific nucleases that are specific for double-stranded DNA. However, some of the phage particles have modified and are not cleaved by the host.
Enzymes are named after the parent organisms from which they are isolated. A fourth letter may be added to represent the strain or serotype of the bacteria. Most enzymes recognize either a sequence of four base pairs (a 4 base cutter) or a sequence of six base pairs (there may be certain exceptions and a few enzymes recognize 5 base pairs or more than 6 base pairs).
While most enzymes have highly specific recognition sequences, some may be relatively less specific. However, the efficiency of ss:DNA digestion as well as the site specificity is much less. It must have the entire coding region of the gene but small enough to be accommodated in the vector.
Other markers that can be used for selection are the presence or absence of some of the metabolic enzymes such as β-galactosidase, glutamine pyruvate transaminase (gpt), thymidine kinase (TK) and dihydrofolate reductase (DHFR). It must have one or more of the marker genes so that differential selection (between recombinant and non-recombinant as well as between transfected and non-transfected cells) can be achieved. In the first type of vectors (the insertion vectors), the foreign DNA is cloned into the non-essential region of the λ genome.
Some of the commonly used vectors are λgt10, λgt11, λcharon vectors, EMBL series of vectors and the λZap vectors. If the sequence (complete or even partial) of the protein encoded by the desired gene is known, it can be used to deduce the nucleic acid sequence of the protein. Since the codon matching index for different organisms is known, it can be used to narrow down the possible sequence of the gene.
To produce the protein, the gene must be cloned in the right orientation and also in the right reading frame. Therefore, it is preferable to use polyclonal antibodies rather than monoclonal antibodies for immunoscreening of gene libraries. Hybrid selection can be used to screen a library if none of the above methods are available to isolate a clone.
But it is one of the final pieces of evidence for determining the identity of clones.
Specific hybridization with related probes
In this method, each clone is used to select the mRNA against which it was synthesized; each mRNA is isolated and translated. To minimize the number of analyses, the clones are collected in groups and analyzed together. This is a very cumbersome process and is not usually preferred for library screening purposes.
Once a clone has been obtained from library screening, it is essential that its identity be established beyond reasonable doubt.
Restriction analysis
Hybrid selection, in vitro translation of selected mRNA and imuno-precipitation of the synthesized protein
Nucleotide sequencing
From a combination of all these analyses, the identity of the clone is identified and characterized. DNA polymerase then synthesizes complementary strands, making two copies of the target sequence. In this process the target sequence is an RNA molecule instead of the usual DNA.
In the first cycle, the ds-DNA is synthesized, which is then amplified to produce multiple copies of the target DNA. This is a method of generating copies of only one of the two strands of the target DNA. However, once one of the primers is exhausted, it can no longer initiate the synthesis of its complementary strand.
Here, single restriction sites are used to cut the template DNA on both sides of the target. It is possible to include restriction sites upstream of the primers that result in amplification of the fragment, together with a suitable restriction site that can be used for cloning. Recombinant insulin, human growth hormone and factor VIII are some of the commercially available recombinant proteins.
Gene therapy is one of the advanced tools for treating a disease caused by a defect or change in genetic material. It involves increasing the function of a defective gene or suppressing an overactive gene. The patient's faulty gene can be corrected by a number of different strategies. a) its replacement by the normal functional copy of the gene (replacement therapy).
Currently, gene augmentation appears to be the method of choice for most gene therapy strategies. The target cell will therefore have a copy of the correct gene, along with the defective gene. A number of different methods have been developed and/or investigated for introducing the functional gene into the patient's cells.
If a natural killer (NK) cell fuses with one of the arms, the resulting molecule can be used to specifically kill the target cell. The fusion mixture of cells (containing fused cells and unfused parental myeloma cells and B cells) is then cultured in the presence of HAT medium.
