Agarose enzymic analysis

A PCR restriction enzyme analysis takes advantage of the occurrence of approximately 40 thalassemia mutations that either introduce or remove a restriction endonuclease site. The PCR amplified target sequence is digested using restriction enzymes (enzymes that cleave DNA at particular nucleotide sequences) and the pattern of fragmentation on an agarose gel defines the presence or absence of a particular mutation. 

Fig. 2. Restriction enzyme analysis and Southern-blot hybridization of human tumor DNA. Human tumor DNA (20 p-g) was isolated (16) and digested with coRI restriction enzyme, electrophoresed on a 0.8% agarose gel (A shows the ethidium-bromide stain) and after Southern transfer, probed with P-labeled pcD-p(ADP-ribose) polymerase cDNA, (B). Lanes 1, Human placental DNA 2, Laryngeal squamous ceU carcinoma (SQ-20B) 3, Lung adenocarcinoma (A549) 4, Cervical carcinoma (HeLaSs) Ewing s sarcoma (A4573) 6, normal human fibroblasts (NHF). Marker-lambda-DNA.

Two isolation procedures based on methods in category 2 are described in this experiment, a large-scale and a microscale method. Each procedure yields plasmid DNA that is sufficiently pure for size analysis by agarose electrophoresis and for digestion by restriction enzymes as described in Experiment 15. 

Restriction endonucleases catalyze the hydrolysis of specific phosphodi-ester bonds in double-stranded DNA. These enzymes are often used to linearize a circular plasmid for hybrid DNA construction. They have also found use in the analysis of DNA and the construction of restriction maps. In this experiment, students will incubate various restriction enzymes with plasmid or viral DNA and analyze the product DNA fragments by agarose gel electrophoresis. 

Reaction mixtures from restriction enzyme digestion may be analyzed directly by agarose gel electrophoresis. This technique combines high resolving power and sensitive detection to allow the analysis of minute amounts of DNA fragments. 

Table 20-3 lists the principal plasma proteins and their half-lives, pi, molecular weights, and preferred method of analysis the individual proteins are listed in the order of their electrophoretic mobilities in agarose gels at pH 8.6. These proteins are described later in this chapter other chapters in this book describe many more proteins enzymes (see Chapter 21) lipoproteins (see Chapter 26) hormones (see Chapter 28) and hemoglobin, fibrinogen, and other coagulation proteins (see Chapter 31). The interim consensus reference intervals for 14 plasma proteins are listed in Table 20-4, pending the publication of more definitive intervals. . 

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