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Ch. 20 - Recombinant DNA Technology
Klug - Concepts of Genetics  12th Edition
Klug12th EditionConcepts of Genetics ISBN: 9780135564776Not the one you use?Change textbook
Chapter 20, Problem 18

To estimate the number of cleavage sites in a particular piece of DNA with a known size, you can apply the formula N/4ⁿ where N is the number of base pairs in the target DNA and n is the number of bases in the recognition sequence of the restriction enzyme. If the recognition sequence for BamHI is GGATCC and the λ\lambda phage DNA contains approximately 48,500 bp, how many cleavage sites would you expect?

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1
Identify the total number of base pairs (N) in the DNA. Here, N = 48,500 bp.
Determine the length of the recognition sequence (n) for the restriction enzyme BamHI. The sequence is GGATCC, which has 6 bases, so n = 6.
Apply the formula to estimate the number of cleavage sites: \( \frac{N}{4^n} \). This means you divide the total base pairs by 4 raised to the power of the recognition sequence length.
Calculate \( 4^n \) by raising 4 to the power of 6, since the recognition sequence is 6 bases long.
Divide the total number of base pairs (48,500) by the value obtained in the previous step to estimate the number of BamHI cleavage sites in the phage DNA.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Restriction Enzyme Recognition Sites

Restriction enzymes recognize specific short DNA sequences, called recognition sites, where they cut the DNA. For example, BamHI recognizes the sequence GGATCC, which is 6 bases long. The frequency of these sites in a DNA molecule depends on the length and sequence specificity of the enzyme.
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Probability of Recognition Site Occurrence

The expected number of recognition sites in a DNA sequence can be estimated using the formula N/4ⁿ, where N is the total number of base pairs and n is the length of the recognition sequence. This formula assumes equal base distribution and random sequence, calculating how often a specific sequence appears by chance.
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Application of the Formula to DNA Size

By applying the formula to a DNA molecule of known size, such as a 48,500 bp phage DNA, and a recognition sequence length of 6 bases (BamHI), you can estimate the number of cleavage sites. This helps predict how many fragments will result from enzyme digestion.
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Related Practice
Textbook Question

You have recovered a cloned DNA segment from a vector and determine that the insert is 1300 bp in length. To characterize this cloned segment, you isolate the insert and decide to construct a restriction map. Using enzyme I and enzyme II, followed by gel electrophoresis, you determine the number and size of the fragments produced by enzymes I and II alone and in combination, as recorded in the following table. Construct a restriction map from these data, showing the positions of the restriction-enzyme cutting sites relative to one another and the distance between them in units of base pairs.

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Textbook Question

To create a cDNA library, cDNA can be inserted into vectors and cloned. In the analysis of cDNA clones, it is often difficult to find clones that are full length—that is, many clones are shorter than the mature mRNA molecules from which they are derived. Why is this so?

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Textbook Question

Although the capture and trading of great apes has been banned in 112 countries since 1973, it is estimated that about 1000 chimpanzees are removed annually from Africa and smuggled into Europe, the United States, and Japan. This illegal trade is often disguised by simulating births in captivity. Until recently, genetic identity tests to uncover these illegal activities were not used because of the lack of highly polymorphic markers (markers that vary from one individual to the next) and the difficulties of obtaining chimpanzee blood samples. A study was reported in which DNA samples were extracted from freshly plucked chimpanzee hair roots and used as templates for PCR. The primers used in these studies flank highly polymorphic sites in human DNA that result from variable numbers of tandem nucleotide repeats. Several offspring and their putative parents were tested to determine whether the offspring were 'legitimate' or the product of illegal trading. The data are shown in the following Southern blot.

Examine the data carefully and choose the best conclusion.

a. None of the offspring is legitimate.

b. Offspring B and C are not the products of these parents and were probably purchased on the illegal market. The data are consistent with offspring A being legitimate.

c. Offspring A and B are products of the parents shown, but C is not and was therefore probably purchased on the illegal market.

d. There are not enough data to draw any conclusions. Additional polymorphic sites should be examined.

e. No conclusion can be drawn because 'human' primers were used.

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Textbook Question

In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges

(a) 92-26 °C

(b) 45-65 °C and

(c) 65-75 °C

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Textbook Question

We usually think of enzymes as being most active at around 37°C, yet in PCR the DNA polymerase is subjected to multiple exposures of relatively high temperatures and seems to function appropriately at 65–75°C. What is special about the DNA polymerase typically used in PCR?

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Textbook Question

Traditional Sanger sequencing has largely been replaced in recent years by next-generation and third-generation sequencing approaches. Describe advantages of these sequencing methods over first-generation Sanger sequencing.

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