Randomly Amplified Polymorphic Dna Rapd

Randomly Amplified Polymorphic DNA, or RAPD, is usually found in the plural as RAPDs and is pronounced "rapids," partly because it is a quick way to get a lot of information about the genes of an organism under investigation. The purpose of RAPDs is to test how closely related two organisms are. In practice, DNA samples from unknown organisms are compared with DNA from a previously characterized organism. For example, traces of blood from a crime scene may be compared to randomly amplified polymorphic DNA (RAPD) Method for testing genetic relatedness using PCR to amplify arbitrarily chosen sequences TA cloning Procedure that uses Taq polymerase to generate single 3'-A overhangs on the ends of DNA segments that are used to clone DNA

into a vector with matching 3'-T overhangs TA cloning vector Vector with single 3'-T overhangs (in its linearized form) that is used to clone DNA segments with single 3'-A overhangs generated by Taq polymerase

FIGURE 23.11 TA Cloning

When Taq polymerase amplifies a piece of DNA during PCR, the terminal transferase activity of Taq adds an extra adenine at the 3' end of the PCR product. The TA cloning vector was designed so that when linearized it has single 5' thymidine overhangs. The PCR product can be ligated into this vector without the need for special restriction enzyme sites.

Terminal transferase activity

OF Taq POLYMERASE

5 Double stranded 3' DNA

PCR may be performed with arbitrary primers. Comparing results from two samples of DNA reveals their relatedness.

possible suspects, or disease-causing microorganisms may be related to known pathogens to help trace an epidemic.

The principle of RAPDs is statistically based. Given any particular five-base sequence, such as ACCGA, how often will this exact sequence appear in any random length of DNA? Since there are four different bases to choose from, one in every 45 (or 4 x 4 x 4 x 4 x 4 = 1,024) stretches of five bases will—on average—be the chosen sequence.Any arbitrarily chosen 11-base sequence will be found once in approximately every 4 million bases. This is approximately the amount of DNA in a bacterial cell. In other words, any chosen 11-base sequence is expected to occur by chance once only in the entire bacterial genome. For higher organisms, with much more DNA per cell, a longer sequence would be needed for uniqueness.

For RAPDs, the arbitrarily chosen sequence should be rare but not unique. PCR primers are made using the chosen sequence and a PCR reaction is run using the total DNA of the organism as a template. Every now and then a primer will find a correct match, purely by chance, on the template DNA (Fig. 23.12). For PCR amplification to occur there must be two such sites facing each other on opposite strands of the DNA. The sites must be no more than a few thousand bases apart for the reaction to work well. The likelihood of two correct matches in this arrangement is quite low.

In practice, the length of the primers is chosen to give five to 10 PCR products. For higher organisms, primers of around 10 bases are typical. The bands from PCR are separated by gel electrophoresis (see Ch. 21) to measure their sizes. The procedure is repeated several times with primers of different sequence. The result is a diagnostic

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