To study genes in the laboratory, it is necessary to have sufficient amount of DNA for analysis, such as determining its sequence, i.e. determining the exact sequence of all the base pairs in the gene. They have their own origin of replication (ori region), and can replicate independently of the host chromosome. They contain origins of replication from M13 or F1 phage and residual features of the vector from plasmids.
BACs have become one of the most widely used vectors for the preparation of genomic libraries. Thus, most plasmid vectors used for cloning in B.subtilis are derived from S.aureus plasmids. It contains the 2 μm origin of replication, the yeast leu2 gene as a selectable marker, and the complete sequence of pBR322. The leu2 gene encodes an enzyme involved in the biosynthesis of the amino acid leucine.
The ends of the insert are also obtained in the same way, so that the foreign DNA is only ligated in one direction (Fig. 8). The problem can be solved by using high concentrations of both DNAs (vector and insert) and of the DNA ligase enzyme. The blunt end of the adapter binds to the DNA insert to produce DNA molecules with sticky ends.
Assembly of the phage particles occurs automatically in the test tube and mature assembled phages are allowed to infect E.coli cells.
Selection of recombinant clones by insertional inactivation of lacz gene
For the expression of cloned genes, special types of vectors called expression vectors are used, which are designed so that a foreign gene can be placed under the control of the host cell's transcriptional and translational machinery. Since the gene is cloned, it is relatively simple to mutate it, obtaining mutant protein, to see how changes in the gene can affect the function of the protein it codes for. Termination sequence: site which marks the point at the end of the gene where transcription stops.
It contains a sequence complementary to the 3' end of the 16S ribosomal RNA, which is part of the 30S ribosomal subunit. Most E.coli expression vectors use either the promoter of the lac or trp operons from E.coli or the λPL promoter responsible for transcription of λ DNA. Such vectors are constructed by joining an appropriate prokaryotic promoter, bacterial Shine Dalgarno sequence and the start codon in such a way that when a foreign gene is cloned these are in front of the gene.
This will enable transcription and translation of the foreign gene and the resulting gene product is a pure protein. The foreign gene is inserted into the coding region of a vector gene in such a way that the product of gene expression is obtained as a hybrid protein consisting of the short peptide (e.g. part of β-galactosidase) encoded by vector gene attached to the amino terminus of foreign protein. This allows export of the recombinant protein either to the culture medium or to the periplasmic space between the inner and outer cell membranes.
These problems may either be due to the sequence of the foreign gene or to the limitations of E.coli as a host for recombinant protein synthesis. a) Problems due to the sequence of the foreign gene. Many variants of expression vectors are currently available for the production of heterologous proteins in yeast and these are derivatives of the YIp, YEp and YCp plasmids. The cloned genes are placed under the control of the galactose-inducible promoter.
PCR works with repeated cycles, each cycle consists of three steps: i) denaturation, i.e. separation of the DNA chain, which takes place at 94 °C for 3-5 minutes, ii) annealing or binding of primers at 50-60 °C for 1-2 minutes. and iii) extension of the charged strands at 72 °C for 2 min. The PCR approach is used when primers are available that anneal to both sides of the gene of interest. Genomic libraries are commonly used to isolate genes when information about the gene product is unknown or when we want to study the regulatory sequences of a gene.
This depends on the size of the cloned DNA fragments and the size of the genome of the organism whose genomic library is constructed. Where N represents the number of clones to be screened, p is the probability of finding a clone, ln represents the natural log, f is the cloned DNA fragment size, and n is the genome size.
CONSTRUCTION OF GENOMIC LIBRARY
Transformation of the host cells: The recombinant vectors containing cDNAs corresponding to mRNAs are introduced into suitable host cells. The cDNA library is then screened to identify the clone carrying the desired gene. This method is used when we know the amino acid sequence of the protein, that is, the product of the gene, and the size of the gene is small.
Using codons for different amino acids, it is possible to deduce the nucleotide sequence of the gene from the sequence of amino acids in the protein. Once the base sequence of the gene is deduced, the polynucleotide with the same base sequence can be chemically synthesized. The mutant phenotype and the position of the mutation in the genetic map indicate that the correct gene is affected.
By using the DNA sequence of the transposon, DNA clones containing the target gene can be identified, consequently the gene is cloned. The identification of a specific clone from a DNA library can be performed by exploiting either the sequence of the clone or the structure/. A probe is a piece of DNA or RNA that contains a portion of the sequence that is complementary to the desired gene we are looking for.
The probe can be labeled radioactive (with P32) or non-radioactive (biotin, digoxigenin and fluorescent dyes, etc.). Probes can be synthesized chemically based on the amino acid sequence of the protein encoded by the gene. A buffer-saturated Whatman No. 1 filter paper is placed on a support such as a glass plate and two edges of the filter are dipped in buffer solution.
The gel is placed on top of the filter paper placed on the support (Fig. 21). A sheet of nitrocellulose membrane, cut to size from the gel, is placed on top of the gel. A stack of dry, rough filter paper the size of the gel is then placed on top of the nitrocellulose filter.
The location of the DNA fragment that hybridizes to the probe can be detected by autoradiography (when the probe is radiolabeled). When restriction enzyme digestion cuts DNA flanking the VNTRs (the flanking sequences are conserved between species), the lengths of the resulting fragments will be variable depending on the number of repeats at a given locus.
