Techniques to Further Analyze Long Stretches of DNA

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DNA that can be inserted between the phage arms. A cosmid vector can accommodate foreign DNA inserts of approximately 45 kb. Yeast artificial chromosomes (YACs) have been developed to clone DNA fragments of 200–500 kb lengths. While cosmid and yeast artificial chromosome vectors are difficult to work with, their libraries permit the cloning of large genes with their flanking regulatory sequences, as well as families of genes or contiguous genes.
Cosmid vectors are a cross between plasmid and bacteriophage vectors. Cosmids contain an antibiotic­resistance gene for selection of recombinant DNA molecules, an origin of replication for propagation in bacteria, and a cos site for packaging of recombinant molecules in bacteriophage particles. The bacteriophage with recombinant cosmid DNA can infect E. coli and inject its DNA into the cell. Cosmid vectors contain only approximately 5 kb of the 50­kb bacteriophage DNA and therefore cannot direct replication and assembly of new infectious phage particles. Instead, the recombinant cosmid DNA circularizes and replicates as a large plasmid. Bacterial colonies with recombinants of interest can be selected and amplified by methods similar to those described for plasmids.
Standard cloning procedures and some novel methods are employed to construct YACs. Very large foreign DNA fragments are joined to yeast DNA sequences, one that functions as a telomere (distal extremity of chromosome arm) and another that functions as a centromere and as an origin of replication. The recombinant YAC DNA is introduced into the yeast by transformation. The YAC constructs are designed so that yeast transformed with recombinant chromosomes grow as visually distinguishable colonies. This facilitates selection and analysis of cloned DNA fragments.
18.10— Techniques to Further Analyze Long Stretches of DNA
Subcloning Permits Definition of Large Segments of DNA
Complete analysis of functional elements in a cloned DNA fragment requires sequencing of the entire molecule. Current techniques can sequence 200–400 bases in a DNA fragment, yet cloned DNA inserts are frequently much larger. Restriction maps of the initial DNA clone are essential for cleaving the DNA into smaller pieces to be recloned, or subcloned for further analysis. The sequences of each of the small subcloned DNA fragments can be determined. Overlapping regions of the subcloned DNA properly align and confirm the entire sequence of the original DNA clone.
Sequencing can often be accomplished without subcloning. Antisense primers can be synthesized that are complementary to the initially sequenced 3 ends of the cloned DNA. This process is repeated until the full length of the cloned DNA has been sequenced. This method obviates the need to prepare subclones but it requires synthesis/purchase of numerous primers. On the other hand, the subcloned DNA is always inserted back into the same region of the plasmid. Therefore one set of primers complementary to the plasmid DNA sequences flanking the inserted DNA can be used for all of the sequencing reactions with subcloned DNA.
Chromosome Walking Is a Technique to Define Gene Arrangement in Long Stretches of DNA
Knowledge of how genes and their regulatory elements are arranged in a chromosome should lead to an understanding of how sets of genes may be coordinately regulated. Currently, it is difficult to clone DNA fragments large enough to identify contiguous genes. The combination of several techniques allows for the analysis of very long stretches of DNA (50–100 kb). The method, chromosome walking, is possible because phage or cosmid libraries contain
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Figure 18.19 Chromosome walking to analyze contiguous DNA segments in a genome. Initially, a DNA fragment is labeled by nick translation to screen a library for recombinant phage carrying a gene of interest. The amplified DNA is mapped with a battery of restriction endonucleases to select a new region (1a) within the original cloned DNA that can be recloned (subcloned). The subcloned DNA (1a) is used to identify other DNA fragments within the original library that would overlap the initially amplified DNA region. The process can be repeated many times to identify contiguous DNA regions upstream and downstream of the initial DNA (gene 1) of interest.
partially cleaved genomic DNA cut at specific restriction endonuclease sites. The cloned fragments will contain overlapping sequences with other cloned fragments. Overlapping regions are identified by restriction mapping, subcloning, screening phage or cosmid libraries, and sequencing procedures.
The overall procedure of chromosome walking is shown in Figure 18.19. Initially the phage library is screened for a sequence of interest with a DNA or RNA probe. The cloned DNA is restriction mapped and a small segment is subcloned in a plasmid, amplified, purified, and labeled by nick translation. This labeled probe is then used to rescreen the phage library for complementary sequences, which are then cloned. The newly identified overlapping cloned DNA is then treated in the same fashion as the initial DNA clone to search for other overlapping sequences. Caution must be taken that the subcloned DNA does not contain a sequence common to the large numbers of repeating DNA