Chapter 5 : Recombinant DNA Technology

Restriction endonucleases recognize and cut specific sites in a DNA molecule. The arrows indicate the cleavage sites of one such endonuclease.

Gel electrophoresis separates DNA fragments by size.

A stained agarose gel showing separated DNA fragments. The outlines of the sample wells are visible at the top of the gel. The lanes at the far right and left contain a mixture of fragments of known size.

DNA ligase joins DNA fragments by forming bonds between the 3′ and 5′ ends of the two backbones.

Hybridization is the formation of base pairs between two complementary single-stranded nucleic acid molecules. The molecules can be the same or different lengths.

Hybridization analysis. A single-stranded probe is added to denatured sample DNA.

A given DNA sequence can be localized to a specific restriction fragment by blotting DNA fragments from an electrophoresis gel to a membrane and conducting hybridization analysis on the membrane.

DNA polymerase makes copies of existing DNA molecules (known as the template). The DNA synthesis reaction requires a primer hybridized to single-stranded template DNA, the enzyme, and all four nucleotides. The primer becomes part of the new DNA molecule.

Reverse transcriptase uses RNA as a template and synthesizes a cDNA copy. Through additional reactions, the RNA can be removed and replaced with a second DNA strand.

PCR produces many copies of a DNA segment lying between and including the sequences at which two single-stranded primers hybridize to the template DNA molecule. The primers are usually synthetic oligonucleotides.

Testing for the presence of a DNA sequence using PCR.

Cloning of DNA in recombinant bacterial plasmids.

DNA libraries.

A home pregnancy test uses antibodies to detect the pregnancy hormone, HCG. (A) The absorbent wick contains dispersed antibody molecules labeled with a colored indicator. (B) To perform the test, the wick is dipped in urine, which migrates up the absorbent material. (C) If the urine contains HCG, the dispersed colored antibody molecules will bind to it and be swept along with the urine. (D) The indicator window sits over a line of molecular traps for the antibody. If HCG-antibody complexes are carried up the wick in the urine, they will be trapped there, forming a colored line under the window. The colored line indicates the presence of HCG and is a positive result.

Isolating E. coli mutants that cannot biosynthesizehistidine (His−).

Using genetics to find an E. coli gene for histidine biosynthesis.

Genetic testing for SSA. bp, base pairs.

An evolutionary tree based on the amino acid sequence of the protein cytochrome c. The numbers represent the numbers of amino acid changes between two nodes of the tree. For example, when the common ancestor of cartilaginous fish diverged from the line leading to bony fish, mammals, etc., it evolved and accumulated two amino acid changes in its cytochrome c protein before the lines leading to dogfish and lamprey diverged. The dogfish line accumulated nine more amino acid changes in becoming the modern organism; the lamprey accumulated eight changes. Thus, the amino acid sequences of cytochrome c in lamprey and dogfish have 17 differences. (From A. Lehninger, D. Nelson, and M. Cox, Principles of Biochemistry, 2nd ed., Worth Publishers, Inc., New York, NY, 1993.)

Neanderthals (skull cast on right), with their large brains and anatomy similar to ours, were long believed to be direct ancestors of modern humans (skull model on left). Mitochondrial DNA analysis suggests that modern humans instead arose independently from Neanderthals and displaced them. (Photograph courtesy of SOMSO Models, Coburg, Germany.)

DNA fingerprinting by RFLP analysis. Radioactive probes are detected by exposing the membrane to X-ray film after hybridization is complete.

Genes are often cloned so that their expression can be controlled. For example, a gene cloned with the lac promoter will be expressed only if lactose is present in the growth medium.

Plant tissue culture. (A) Pieces of plant tissue can be induced to form undifferentiated callus cells (plates on right). Manipulation of hormones in the growth medium causes the callus to differentiate into tiny plantlets (plates on left). (B) Close-up of plantlet. Note the fuzzy roots and tiny leaves. (Photographs courtesy of Syngenta.)

Making a transgenic mouse with ES cells.