Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Construction and Analysis of Genomic Libraries
Isolation of Genes

There are three main approaches to obtaining target genes: isolation from genomic DNA, chemical-enzymatic synthesis (in vitro), and METABOLISM/31.html">Transcription of cellular Messenger RNA using Reverse Transcriptase (RNA-dependent DNA polymerase, or revertase).

Gene isolation from DNA. Most Introduction/32.html">Genetic Engineering techniques are based on cutting specific fragments out of DNA molecules and joining them with other fragments to produce recombinant (chimeric) DNA. In this process, DNA isolated from Cells is fragmented using Enzymes capable of cleaving DNA at strictly specific sites. These enzymes are Restriction Endonucleases (restriction enzymes), the General characteristics of which are discussed in Chapter 2. Genetic engineering utilizes restriction enzymes that generate single-stranded (sticky) ends in DNA by staggered cuts, as well as those that form double-stranded (blunt) ends by cleaving right through the center of a recognized nucleotide pair site. An example of the first type of restriction enzyme, which produces sticky ends, is Eco RI (Fig. 2.1), whereas Hind II serves as an example of the second type.

The formation of sticky ends during DNA Cleavage offers the advantage of allowing the resulting fragments to reassociate via homopolymeric sticky ends containing complementary NUCLEOTIDES. This makes it possible to form associations from fragments belonging to different DNA molecules, which forms The basis of most genetic engineering manipulations for producing recombinant DNA (Fig. 20.1). Spontaneously formed associations can be converted into complete molecules by "sealing" them using DNA ligases.

It should be noted that under normal conditions, sticky ends within a single molecule can be held relative to one another by Hydrogen Bonds between complementary bases. However, complementary strands are easily separated by slightly heating DNA solutions (DNA Denaturation). Upon cooling, sticky ends hybridize again through the restoration of hydrogen bonds, provided THE PRINCIPLE OF complementarity is maintained (annealing). As a result of annealing a set of fragments obtained by treating different DNA molecules with the same restriction enzyme, both the original DNA molecules and their hybrids—recombinant DNA—can be formed.

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Fig. 20.1. Schematic representation of recombinant DNA formation upon cleavage of different DNA molecules by restriction enzymes that expose sticky ends in the fragments

In 1972, J. Mertz and R. Davis carried out the first such experiment at Stanford. It later turned out that not all genomes contain restriction sites for the enzymes being used. This applies particularly to small molecules such as Plasmids and phages, which are so small that they contain only a few restriction sites for a limited number of restriction enzymes. This circumstance significantly limits the potential of the method, and therefore a methodology was developed for introducing DNA fragments containing the necessary restriction sites into The Genome.

To achieve this, artificially synthesized blunt-ended oligonucleotide DNAs known as linkers are employed. Linkers are synthesized in such a way that they contain known restriction sites (which requires prior determination of The nucleotide sequence at these sites). DNA intended for linker incorporation is cleaved with one of the restriction enzymes that generate blunt ends, and the resulting fragments are subsequently ligated to the linkers using DNA ligases (Fig. 20.2). An alternative method for obtaining blunt ends is the mechanical shearing of large DNA molecules by rapid stirring of the solution or by forcing it through a narrow aperture. As a result of these manipulations, DNA fragments acquire restriction sites within the added sequences (linkers).

Now the resulting DNA fragment containing the required restriction sites can be joined to another DNA molecule (for example, a vector) treated with the same restriction enzyme, or converted into a circular form by ligating mutually complementary ends.

The described Methods for isolating genes within DNA fragments using restriction enzymes are widespread, but they have several drawbacks. First, it is not always possible to select restriction enzymes that allow the precise excision of the DNA region containing the gene of interest. Second, the excised DNA fragment may contain sequences that hinder the subsequent use of the gene, such as introns in eukaryotic genes. In this case, recombinant DNA cannot be expressed in Prokaryotic Cells, because the latter lack The ability to perform splicing (Chapter 3).

Fig. 20.2. Attachment of linkers containing restriction sites (in this case for the Eco RI restriction enzyme, indicated by arrows) to blunt-ended DNA fragments

Chemical-enzymatic gene synthesis. This method has been used to synthesize and subsequently clone genes determining The Structure of Hormones such as Insulin and Somatostatin, as well as human leukocyte interferon. The Synthesis of the interferon gene was carried out in the USSR in 1984 under the guidance of Academician M.N. Kolosov.

The Essence of the method is as follows: short (8–16 nucleotides) single-stranded DNA fragments are chemically synthesized in vitro and then joined using ligases and annealed (allowing double-stranded DNA molecules to form). This method requires knowing the nucleotide sequence of the gene, since synthesis is carried out without a template. The sequence is usually deduced from the Amino Acid Sequence of the corresponding polypeptide; however, due to the degeneracy of The Genetic Code, determining the true nucleotide sequence of the gene directly is impossible. The true STRUCTURE OF THE gene can be established by DNA Sequencing, but this requires isolating and cloning the corresponding gene first.

The stage of oligonucleotide chemical synthesis is currently fully automated. The method is based on the specific Protection of the 5' or 3' end of a mono- or oligonucleotide to prevent it from participating in Chemical Reactions. If necessary, the blocking groups used for modification can be removed by acid or alkaline Treatment. The Chemical DNA Synthesis cycle includes nucleotide Condensation, removal of the blocking group, and subsequent condensation. A Modification of the method involves attaching the first nucleotide to a solid support and adding subsequent nucleotides one by one after washing the support at each such stage.

Rejoining single-stranded fragments using ligases requires phosphorylation of the 5' ends, which is accomplished using the enzyme polynucleotide kinase and ATP. Single-stranded DNAs can be converted into double-stranded ones either by annealing with a complementary antiparallel strand that has also been chemically synthesized, or by extending the complementary strand using an enzyme (DNA polymerase I is typically used). By combining chemical synthesis and enzymatic steps, for example, an insulin gene 514 bp in length was reconstructed from 66 short synthetic fragments.

Enzymatic gene synthesis. There are two fundamentally different approaches to the enzymatic synthesis of DNA. One of them does not require a template and proceeds according to a program set by the experimenter. Such synthesis is catalyzed by the bacterial enzyme polynucleotide phosphorylase, which is specific for ribonucleotides but is also capable of polymerizing DNA chains at a lower rate. This type of synthesis requires a primer comprising at least 3 nucleotides. Polymerization reactions of this kind have certain limitations and are difficult to control.

The other method of enzymatic synthesis involves The Use of a template, which in The First stage is mRNA isolated from The Cell. Virtually all eukaryotic mRNAs have a "tail" (polyadenylate sequence) at the 3' end. This region is used to generate a primer for the complementary DNA strand: short sequences consisting of thymidylates are added to the mRNA and hybridize with the polyadenylates via annealing (Fig. 20.3). In the presence of a pool of deoxynucleotides, reverse transcriptase catalyzes their addition to the primer in a sequence determined by the mRNA, resulting in the formation of a double-stranded RNA-DNA hybrid. For reasons not yet fully understood, the newly synthesized DNA strand forms a hairpin loop at the end (Fig. 20.3), which occurs only during the in vitro reaction (presumably because the enzyme "turns back"). This hairpin serves as a primer for the synthesis of the second DNA strand. In the next stage, the RNA is degraded using ribonucleases or alkaline Hydrolysis, and the remaining single-stranded DNA with its hairpin is used as a template for synthesizing the second DNA strand (using DNA polymerase I). In The final stage, the hairpin is cleaved using S1 nuclease, which specifically hydrolyzes single-stranded regions of Nucleic Acids. This yields double-stranded "complementary" DNA, or cDNA (the name reflects its defining feature: complementarity to mRNA).

Fig. 20.3. Formation of cDNA during enzymatic synthesis based on mRNA

There are modifications of the described method that avoid many of its drawbacks, particularly the synthesis of incomplete RNA copies (especially in the case of long mRNAs). One variation of the method involves the synthesis of cDNA directly on the vector.