DNA copies

Complementarity between A and T and G and C suggests a mechanism for copying DNA. This is called replication and is discussed in Section 28.10. 

A solution to the problem of introns is to isolate mRNA extracted from the human pancreas cells that make insulin. These cells are rich in insulin mRNA from which introns have already been spliced out. Using the enzyme reverse transcriptase it is possible to convert this spliced mRNA into a DNA copy. This copy DNA (cDNA), which carries the uninterrupted genetic information for insulin can be cloned. Although yeast cells (Saccharomyces) can splice out introns it is normal practice to eliminate them anyway by cDNA cloning. 

Polymerase chain reaction (PCR) is one of the most important techniques for rapid bacterial identification. It consists of repeated cycles of enzymatic reactions in a thermal cycler (PCR machine) that copies DNA strands many times. The DNA amplified in one PCR cycle is used as a template for the next cycle. This results in an exponential increase of the desired target... 

DNA polymerase—Protein that copies DNA by linking together bases that are lined up and paired with complementary bases in a piece of DNA. 

PCR uses natural enzymes to copy DNA molecules. Cells replicate their DNA when they divide, so that each daughter cell will have its own copies. The enzyme, called DNA polymerase, works by attaching to a single strand of DNA and then catalyzing the reactions that synthesize the complementary strand, which joins the other stand to make a double-stranded helix. 

Messenger RNA is isolated from the tissue source selected to contain the Ab-secreting cells. Spleen, bone marrow, tonsil, and lymph node have all been successfully used as a source Copy DNA (cDNA) is then generated by reverse transcription and Fv or Fab regions amplified by PCR. 

Single-copy DNA. A region of the genome the sequence of which is present only once per haploid complement. 

Polymerase chain reaction Fast technique used to copy DNA. 

These enzymes copy DNA sequences by using one strand as a template. The reaction catalyzed by DNA polymerases is the addition of deoxyribonucleotides to a DNA chain by using dNTPs as substrates, as shown in Figure 8-7. 

All DNA polymerases require a template strand, which is copied. DNA polymerases also require a primer, which is complementary to the template. The reaction of DNA polymerases is thus better understood as the addition of nucleotides to a primer to make a sequence complementary to a template. The requirement for template and primer are exactly what would be expected of a replication enzyme. Because DNA is the information store of the cell, any ability of DNA polymerases to make DNA sequences from nothing would lead to the degradation of the cell s information copy. 

When isolated from bacteria, prokaryotic RNA polymerase has two forms The core enzyme and the holoenzyme. The core enzyme is a tetramer whose composition is given as 0C2PP (two alpha subunits, one beta subunit, and one beta-prime subunit). Core RNA polymerase is capable of faithfully copying DNA into RNA but does not initiate at the correct site in a gene. That is, it does not recognize the promoter specifically. Correct promoter recognition is the function of the holoenzyme form of RNA polymerase. 

If the restriction fragment used as primer is large (>100-200 nucleotides) it is necessary to cleave this from the radioactive copy DNA before gel electrophoresis. This reaction usually employs the same restriction enzyme that was used to generate the primer. 

This reaction is frequently useful in resolving pile-ups caused by regions of local secondary structure in the copied DNA (cf section 4.7). 

Enzymatic replication of DNA is a complex process and requires the cooperation of some 20 or more proteins. Arthur Komberg and his colleagues first discovered an enzyme in E. coli that catalyzed the polymerization of deoxyribonucleotides under the direction of a DNA template. This enzyme, DNA polymerase I (Pol I), is now known to be only one of a group of similar enzymes that can copy DNA or RNA templates or both. Three distinct enzymes (Pol I, Pol II, and Pol III) have been isolated from bacterial cells (Table 11.1). These enzymes catalyze the stepwise addition of deoxyribonucleotide residues to the free 3 -hydroxyl end of a preexisting DNA or RNA primer strand thus the enzymatic elongation proceeds in the 5 —> 3 direction. The overall reaction is... 

Single-copy DNA hybridization techniques have seen widespread application to problems in systematics. Most notably, Sibley and Ahlquist1 have produced a phylogeny for many of the birds of the world. Other taxa... 

Given these constraints that impinge on the reassociation of DNA, experimental designs must be constructed that facilitate (1) the separation of single-copy DNA to be used as tracer DNA from repeated sequences and (2) the hybridization of single-copy tracer DNA with driver DNA from the same and different species. 

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