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Chapter: Microbiology and Immunology: Genetic Engineering and Molecular Methods

Genetic Engineering

Genetic engineering is the application of science to social needs. In recent years, engineering based on bacterial genetics has transformed biology. Specified DNA fragments can be isolated and amplified, and their genes can be expressed at high levels.

Genetic Engineering

Genetic engineering is the application of science to social needs. In recent years, engineering based on bacterial genetics has transformed biology. Specified DNA fragments can be isolated and amplified, and their genes can be expressed at high levels. The nucleotide specificity, required for cleavage by restriction enzymes, allows fragments containing genes or parts of genes to be covalently bound to plasmids (vectors) that can then be inserted into bacterial hosts.

      Bacterial colonies or clones carrying specified genes are iden-tified by hybridization of DNA or RNA with chemical or radio-chemical probes. Alternatively, protein products encoded by the genes are recognized either by enzyme activity or by immuno-logic techniques. Thus, genetic engineering techniques are used to isolate virtually any gene with a biochemically recognizable property.

Preparation of DNA Fragments with Restriction Enzymes

The genetic diversity of bacteria is reflected in their remarkable range of restriction enzymes, which possess remarkable selectivity that allows them to recognize specific regions of DNA for cleav-age. DNA sequences recognized by restriction enzymes are pre-dominantly palindromes (inverted sequence repetitions). GAATTC is a typical sequence palindrome, recognized by the frequently used restriction enzyme EcoRI. The inverted repetition, inherent in the complementarity of the G–C and A–T base pairs, results in the 59 sequence TTC being reflected as AAG in the 39 strand.

Most restriction enzymes recognize 4, 6, or 8 base sequences; however, other restriction enzymes recognize 10, 11, 12, or 15 base sequences. Restriction enzymes that recognize 8 bases produce fragments with a typical size of 64,000 bp and are useful for anal-ysis of large genetic regions. Restriction enzymes that recognize more than 10 bases are useful for construction of a physical map and for molecular typing by pulse-field gel electrophoresis.

Physical Separation of Differently Sized DNA Fragments

Much of the simplicity underlying genetic engineering techniques lies in the fact that gel electrophoresis permits DNA fragments to be separated on the basis of size. The smaller the fragment, the more rapid the migration. Overall rate of migra-tion and optimal range of size for separation are determined by the chemical nature of the gel and by the degree of its cross-linking. Highly cross-linked gels optimize the separation of small DNA fragments. The dye ethidium bromide forms a brightly fluorescent color as it binds to DNA, and so small amounts of separated DNA fragments can be photographed on gels. Specific DNA fragments can be recognized by probes containing complementary sequences.

Pulsed-field gel electrophoresis allows the separation of DNA fragments containing up to 100,000 bp (100 kilobase pairs, or kbp). Characterization of such large fragments has allowed construction of a physical map for the chromosomes from several bacterial species.

Enzymes for Dicing, Splicing, and Reversing Nucleic Acids

Restriction endonucleases: The polynucleotide strands ofDNA can also be clipped crosswise at selected positions by means of enzymes called restriction endonucleases. These enzymes recognize foreign DNA and are capable of digesting or hydrolyzing DNA bonds. Presence of enzyme in the bacterial cell protects bacteria against the incompatible DNA of bacte-riophages or plasmids.

In a laboratory, restriction endonuclease enzymes can be used to cleave DNA at desired sites and are a must for the techniques of recombinant DNA technology. So far, hundreds of restric-tion endonucleases have been discovered in bacteria. Each type has a known sequence of 4–10 bp as its target, so sites of cutting can be finely controlled. Endonucleases are named by combin-ing the first letter of the bacterial genus, the first two letters of the species, and the endonuclease number. For example, EcoRI is the first endonuclease found in Escherichia coli and HindIII is the third endonuclease discovered in Haemophilus influenzae typed.

Restriction fragment length polymorphisms: The pieces ofDNA produced by restriction endonucleases are termed restric-tion fragments.Because genomes of members of the same spe-cies can vary in the cutting pattern by specific endonucleases, it is possible to detect genetic differences by restriction fragmentlength polymorphisms (RFLPs).

Hundreds of cleavage sites that produce RFLPs are dis-tributed throughout genomes. Because RFLPs serve as a type of genetic marker, they can help locate specific sites along a DNA strand. The RFLPs are thus useful in preparation of gene maps and DNA profiles, and also in analysis of genetic relationships.

Ligase: It is an enzyme necessary to seal the sticky ends togetherby rejoining the phosphate–sugar bonds cut by endonucleases. Its main application is in final splicing of genes into plasmids and chromosomes.

Reverse transcriptase: It is an enzyme, best known for its rolein the replication of the AIDS virus and other retroviruses. This enzyme is used by geneticists as a valuable tool for converting RNA into DNA.

Complementary DNA: The copies calledcomplementary DNA,or cDNA, can be made from messenger, transfer, ribosomal, and other forms of RNA. The technique provides a valuable means of synthesizing eukaryotic genes from mRNA transcripts. The advantage is that the synthesized gene will be free of the inter-vening sequences (introns) that can complicate the management of eukaryotic genes in genetic engineering. Complementary DNA can also be used to analyze the nucleotide sequence of RNAs, such as those found in ribosomes and transfer RNAs.

Methods Used to Size, Synthesize, and Sequence DNA

The relative sizes of nucleic acids are usually known by the number of base pairs or nucleotides they contain. For example, the palindromic sequences recognized by endonucleases are usually 4–10 bp in length. An average gene in E. coli is approxi-mately 1300 bp, or 1.3 kilobases (kb), and its entire genome is approximately 4.7 million base pairs (Mb). The Epstein–Barr virus, cause of infectious mononucleosis, has a gene of 172 kb. Humans have approximately 3.5 billion base pairs (Bbp) arrayed along 46 chromosomes.

Oligonucleotides are very short pieces of DNA or RNA.Theyvary in length from 2 to 200 bp, although the most common ones are about 20–30 bp. They can be isolated from cells or prepared tailor-made by a DNA synthesizer that limits the length to about 200 nucleotides.

Hybridization probes are used routinely in the cloning of DNA. The amino acid sequence of a protein is used to deduce the DNA sequence from which a probe may be constructed and employed to detect a bacterial colony containing the cloned gene. cDNA, encoded by mRNA, is used to detect the gene that encodes the mRNA.

Types of hybridization

Hybridization is of the following types:

      Northern blot: Hybridization of DNA to RNA is knownas Northern blot, which provides quantitative information about RNA synthesis.

      Southern blot: Hybridization of DNA to DNA is known asSouthern blot. This method is useful to detect specific DNAsequences in restriction fragments separated on gels. These blots can be used to detect overlapping restriction fragments.

      Western blot: It is a technique used for detection of genes,in which antibodies are used to detect cloned genes by bind-ing to their protein products.

Cloning of these fragments makes it possible to isolate flank-ing regions of DNA by a technique known as chromosomalwalking.

DNA sequencing

DNA sequencing shows gene structure that helps research workers to find out the structure of gene products.

Maxam–Gilbert technique and Sanger (dideoxy termination) method are two methods used routinely for DNA sequence determination. Maxam–Gilbert technique depends on the relative chemical liability of different nucleotide bonds, whereas the Sanger method interrupts elongation of DNA sequences by incorporating dideoxynucleotides into the sequences.

Shotgunning:The study of biology has been revolutionized bythe development of technology that allows sequencing and analysis of entire genomes ranging from viruses to unicellular prokaryotic and eukaryotic microorganisms to humans. This has been facilitated by use of the procedure known as shotgunning. In this procedure, the DNA is broken into randomsmaller fragments to create a random fragment library.

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