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    for both questions, assume that the four types of nucleotides are equally likely to be found in dna. calculate how often, on average, a type ii restriction endonuclease is expected to cut a dna molecule if the recognition sequence for the enzyme has 5 bp.

    James

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    get for both questions, assume that the four types of nucleotides are equally likely to be found in dna. calculate how often, on average, a type ii restriction endonuclease is expected to cut a dna molecule if the recognition sequence for the enzyme has 5 bp. from EN Bilgi.

    Problem Set 5 Answers

    1.a. Then

    Eco RI and Hin dIII have a six-base recognition site, so they will cut once per every 46, or 4096 bases.

    b. To determine the number of sites, divide the number of bases in the DNA by the average length of the restriction fragments resulting from the given enzyme.   The size of l is 48,502 base pairs, so the number of expected sites are:

    Eco RI

    Hin dIII

    Hin dII

    Hpa II

    Expected (A+T=C+G)

    b. The cDNA in pBR322 will only consist of the two exons, which are flanked at the ends by EcoRI.  When cut by EcoRI, the cDNA will yield 0.5, 1.5, and 2 kilobase fragments.  Digesting with both EcoRI and HindIII, will yield 0.5, 1, and 1.5 kilobase fragments.

    e. Because the length of the PvuPvuII

             2.5          1.5   0.5              4.5    

    c. Two methods that can be used to labed cDNAs are primer extension and nick translation.  Primer extension uses an oligonucleotide primer and Klenow fragment to synthesize a labeled DNA strand.  In nick translation, a small amount of DNase is used to create nicks in the dDNA.  Labeled oligonucleotides are then added with DNA Polymerase I in order to fill in at the nicks.

             2 kb             2 kb                   3 kb              1 kb          2 kb      

    8. Reverse transcription is used to make cDNA from mRNA, and PCR is used to amplify it. 

    9. a. i) Bam HI    ii) Hin dIII    iii) Eco RI    (iv) Hin dIII and Eco Ri?

    b. Bam

    Problem Set 5 Answers

    Source : www.columbia.edu

    Type II Restriction Enzymes: What You Need to Know

    Read about Type II restriction enzymes and the distinguishing properties of the four principle subtypes.

    Type II Restriction Enzymes: Subtypes, naming conventions, and properties

    What are Type II Restriction Enzymes used for?

    Type II restriction enzymes are the familiar ones used for everyday molecular biology applications such as gene cloning and DNA fragmentation and analysis. These enzymes cleave DNA at fixed positions with respect to their recognition sequence, creating reproducible fragments and distinct gel electrophoresis patterns. Over 3,500 Type II enzymes have been discovered and characterized, recognizing some 350 different DNA sequences. Thousands more ‘putative’ Type II enzymes have been identified by analysis of sequenced bacterial and archaeal genomes, but remain uncharacterized.

     How are Type II Restriction Enzymes named?

    Restriction enzymes are named according to the micro-organism in which they were discovered. The restriction enzyme ‘HindIII’, for example, is the third of several endonuclease activities found in the bacterium Haemophilus influenzae serotype d. The prefix ‘R.’ is added sometimes to distinguish restriction enzymes from the modification enzymes with which they partner in vivo. Thus, ‘R.HindIII’ refers specifically to the restriction enzyme, and ‘M.HindIII’ to the modification enzyme. When there is no ambiguity, the prefix ‘R.’ is omitted.

    Type II Restriction Enzymes Properties

    Type II restriction enzymes are very diverse in terms of amino acid sequence, size, domain organization, subunit composition, co-factor requirements and modes of action. They are loosely classified into a dozen or so sub-types according to their enzymatic behavior. This is a practical classification that reflects their properties rather than their phylogeny. It does not necessarily reflect evolutionary or structural relationships, and the subtypes are not mutually exclusive. An enzyme can belong to several subtypes if it exhibits each of their defining characteristics. We discuss these subtypes in their order of importance; the four principal ones are Type IIP, IIS, IIC, and IIT.

    Type IIP (‘Palindromic’ specificity; one domain)

    Type IIS (‘Shifted cleavage’; two domains)

    Type IIC (Combined ‘restriction-and-modification’ enzymes; three domains)

    Type IIT (two different catalytic sites; heterodimers)

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    Type II Restriction Enzymes: What You Need to Know

    Source : international.neb.com

    restriction enzyme

    Restriction enzyme, protein produced by bacteria that cleaves DNA at specific sites. In bacteria, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes are used in the laboratory to manipulate DNA fragments. Learn about the types and uses of restriction enzymes.

    states of matter

    phase, in thermodynamics, chemically and physically uniform or homogeneous quantity of matter that can be separated mechanically from a nonhomogeneous mixture and that may consist of a single substance or a mixture of substances. The three fundamental phases of matter are solid, liquid, and gas (vapour), but others are considered to exist, including crystalline, colloid, glassy, amorphous, and plasma phases. When a phase in one form is altered to another form, a phase change is said to have occurred.

    General considerations

    A system is a portion of the universe that has been chosen for studying the changes that take place within it in response to varying conditions. A system may be complex, such as a planet, or relatively simple, as the liquid within a glass. Those portions of a system that are physically distinct and mechanically separable from other portions of the system are called phases.

    phase change

    A person's hand pouring blue fluid from a flask into a beaker. Chemistry, scientific experiments, science experiments, science demonstrations, scientific demonstrations.

    Phases within a system exist in a gaseous, liquid, or solid state. Solids are characterized by strong atomic bonding and high viscosity, resulting in a rigid shape. Most solids are crystalline, inasmuch as they have a three-dimensional periodic atomic arrangement; some solids (such as glass) lack this periodic arrangement and are noncrystalline, or amorphous. Gases consist of weakly bonded atoms with no long-range periodicity; gases expand to fill any available space. Liquids have properties intermediate between those of solids and gases. The molecules of a liquid are condensed like those of a solid. Liquids have a definite volume, but their low viscosity enables them to change shape as a function of time. The matter within a system may consist of more than one solid or liquid phase, but a system can contain only a single gas phase, which must be of homogeneous composition because the molecules of gases mix completely in all proportions.

    states of matter

    System variables

    Systems respond to changes in pressure, temperature, and chemical composition, and, as this happens, phases may be created, eliminated, or altered in composition. For example, an increase in pressure may cause a low-density liquid to convert to a denser solid, while an increase in temperature may cause a solid to melt. A change of composition might result in the compositional modification of a preexisting phase or in the gain or loss of a phase.

    The phase rule

    The classification and limitations of phase changes are described by the phase rule, as proposed by the American chemist J. Willard Gibbs in 1876 and based on a rigorous thermodynamic relationship. The phase rule is commonly given in the form P + F = C + 2. The term P refers to the number of phases that are present within the system, and C is the minimum number of independent chemical components that are necessary to describe the composition of all phases within the system. The term F, called the variance, or degrees of freedom, describes the minimum number of variables that must be fixed in order to define a particular condition of the system.

    Phase diagrams

    Unary systems

    Phase relations are commonly described graphically in terms of phase diagrams (see Figure 1). Each point within the diagram indicates a particular combination of pressure and temperature, as well as the phase or phases that exist stably at this pressure and temperature. All phases in Figure 1 have the same composition—that of silicon dioxide, SiO2. The diagram is a representation of a one-component (unary) system, in contrast to a two-component (binary), three-component (ternary), or four-component (quaternary) system. The phases coesite, low quartz, high quartz, tridymite, and cristobalite are solid phases composed of silicon dioxide; each has its own atomic arrangement and distinctive set of physical and chemical properties. The most common form of quartz (found in beach sands and granites) is low quartz. The region labeled anhydrous melt consists of silicon dioxide liquid.

    Different portions of the silicon dioxide system may be examined in terms of the phase rule. At point A a single solid phase exists—low quartz. Substituting the appropriate values into the phase rule P + F = C + 2 yields 1 + F = 1 + 2, so F = 2. For point A (or any point in which only a single phase is stable) the system is divariant—i.e., two degrees of freedom exist. Thus, the two variables (pressure and temperature) can be changed independently, and the same phase assemblage continues to exist.

    Point B is located on the boundary curve between the stability fields of low quartz and high quartz. At all points along this curve, these two phases coexist. Substituting values in the phase rule (2 + F = 1 + 2) will cause a variance of 1 to be obtained. This indicates that one independent variable can be changed such that the same pair of phases will be retained. A second variable must be changed to conform to the first in order for the phase assemblage to remain on the boundary between low and high quartz. The same result holds for the other boundary curves in this system.

    Point C is located at a triple point, a condition in which three stability fields intersect. The phase rule (3 + F = 1 + 2) indicates that the variance is 0. Point C is therefore an invariant point; a change in either pressure or temperature results in the loss of one or more phases. The phase rule also reveals that no more than three phases can stably coexist in a one-component system because additional phases would lead to negative variance.

    restriction enzyme

    Source : www.britannica.com

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