Gel Electrophoresis - uni-jena.de .1 Analytische Chemie I Gel Electrophoresis Separating DNA and

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Text of Gel Electrophoresis - uni-jena.de .1 Analytische Chemie I Gel Electrophoresis Separating DNA and

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    Analytische Chemie I

    Gel Electrophoresis

    Separating DNA and Proteins

    Supervisors

    Charles Vidoudez, charles.vidoudez@uni-jena.de, 03641-948178

    Matthew Welling, matthew.welling@uni-jena.de, 03641-948178

    IAAC, Lehrstuhl fr instrumentelle Analytik, Lessingstr. 8, Raum 306, 07743 Jena

    At the beginning of the practical the theoretical introduction will be discussed. Please consider the

    following control questions:

    What are the major differences between agarose gels and polyacrylamide gels? What are their

    advantages and disadvantages?

    Which classes of molecules can be run on each type of gel?

    Which forces are influencing the molecules during their migration in the gels?

    What is the concept of restriction fragment analyses?

    How can large quantities of proteins of interest be obtained?

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    1. From the genetic code to proteins

    All living cells on Earth store their information in the form of double-stranded molecules of DNA, which

    provide the blueprints for proteins. In order to understand this code of life analytical methods were

    developed that allow separation of these biomacromolecules. This lab class addresses the fundamental

    methods currently employed for DNA and protein separation.

    1.1. Base pairs

    There are four heterocyclic bases which are found in deoxyribonucleic acid (DNA): two purine bases,

    adenine (A) and guanine (G), and two pyrimidine bases, thymine (T) and cytosine (C). Each base can pair

    up specifically with another base adenine with thymine (AT) and guanine with cytosine (GC). This

    base pairing is facilitated by two or three hydrogen bonds.

    1.2. DNA

    When bound to a sugar and a phosphate, the bases form a nucleotide. A single strand of DNA is a

    polymer consisting of nucleotide monomers joined together by sugar-phosphate linkages. Each polymeric

    strand of DNA coils up into a helix and is bonded to another complimentary strand by hydrogen bonds

    between the bases. The resulting double stranded helix of DNA can adopt either a linear or circular shape

    and is usually described in terms of the number of base pairs (bp, 1000bp = kb).

    1.3. Gene

    The instructions stored within every living cell are its genes. A gene is defined as the segment of DNA

    sequence corresponding to the production of a single protein (or single catalytic or structural RNA

    molecule). Other parts of the DNA sequence can display structural purposes or are involved in regulating

    Figure 1: the base pairs

    Figure 2: the DNA helix

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    the use of the genetic information. However, there are still sequences whose purposes have not yet been

    discovered.

    1.4. RNA

    During transcription, the sequence of the gene is copied into a ribonucleic

    acid (RNA) nucleotide sequence. The RNA is complimentary to the DNA

    sequence but only exists as a single strand. This then instructs the cell

    machinery on protein synthesis (translation) using three nucleotide codes

    to indicate different amino acids.

    1.5. Proteins

    Proteins are partly structuralas in connective tissueand partly

    functionalas in enzymes, the catalysts for biological reactions. Each cell contains many different

    protein molecules which, aside from water, make up for most of its mass. The structure of proteins may

    be divided into four levels of organisation.

    Primary structure: a simple amino acid sequence linked by peptide bonds.

    Secondary structure: The folding of parts of the primary structure into either helices or sheets.

    Tertiary structure: The full 3D organisation of the polypeptide chain.

    Quaternary structure: The complete structure of a protein with more than one polypeptide chain.

    The destruction or rearrangement of the quaternary and tertiary, and in some cases the secondary,

    structure is known as denaturation.

    2. Gel Electrophoresis, introduction

    A molecule with a net charge will migrate in an electric field. This phenomenon, termed electrophoresis,

    offers a powerful means of separating macromolecules, such as proteins, DNA and RNA. The velocity of

    migration (v) of a molecule in an electric field depends on the electric field strength (E) and on the

    electrophoretic mobility () of the molecule (Equation 1).

    The electrophoretic mobility is a parameter unique for each molecule and each medium. Electrophoretic

    separation is nearly always carried out in gels due to their ability to serve as a molecular sieve that

    enhances separation by modifying . Molecules that are small compared to the pores in the gel readily

    move through the gel, whereas molecules much larger than the pores are almost immobile. Intermediate-

    size molecules move through the gel with various degrees of facility.

    In the case of gel electrophoresis, the following relation is found:

    where 0 is the free electrophoretic mobility of the molecule (mobility in a non-sieving medium), Kr the

    retardation coefficient and the concentration of the gel. 0 is dependant on the mass-to-charge ratio of

    the molecule, whereas Kr is related to the propriety of the gel, the size and the shape of the migrating

    molecule.

    The matrix used for gel electrophoresis is either a cross-linked polymer such as polyacrylamide, or a

    linear polymer such as agarose.

    Figure 3: from gene to protein

    v E=Equation 1:

    0log log rK = Equation 2:

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    3. Agarose Gel Electrophoresis

    3.1. Application

    Agarose gels are the standard method used to separate, identify and purify DNA (and RNA). Their

    resolving power is lower than polyacrylamide gels but the range of separation is greater and they are

    easier to prepare. DNA fragments ranging from 200bp up to 50kb can be separated. They can also be used

    as a tool to isolate a defined DNA fragment or as a preparative step in southern blot (DNA specific

    detection) or northern blot (RNA specific detection).

    3.2. Apparatus

    The most commonly used configuration is a horizontal slab gel. The gel is poured on a glass or plastic

    tray that can be installed on a platform in the electrophoresis tank. Electrophoresis is carried out with the

    gel submerged just beneath the surface of a buffer. The resistance of the gel to electric current is almost

    the same as that of the buffer, and so a considerable fraction of the applied current passes along the length

    of the gel (Figure 4).

    Samples are inserted into wells placed at one end of the gel. In the buffer (pH = 7.5-7.8) DNA is

    negatively charged due to deprotonation of the phosphate linkage and will migrate towards the cathode.

    3.3. The Gel

    3.3.1. Structure of the Gel

    Agarose, which is extracted from seaweed, is a linear polymer whose basic structure is based on the

    alternate structure of -D-galactose units and 3,6-anhydro--L-galactose units (Figure 5). The linear

    strains form double helices, which are extensively aggregated. These aggregations provide the structural

    frame for the gel network. A typical gel contains 0.5-2% of agarose.

    3.3.2. Preparation

    Agarose powder is melted in a buffered solution by boiling. The solution is poured into a gel tray and

    polymerisation occurs as it cools to room temperature.

    - + Agarose Gel

    Platinum Electrodes

    DNA migration

    Figure 4: Agarose Gel Electrophoresis apparatus a. sideview b. top down view.

    The row of wells allows samples to be run in parallel, including a standard ladder.

    Buffer Well

    +

    -

    a b

    DNA m

    igration

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    3.4. Factors affecting the rate of DNA migration

    Different factors affect the velocity of DNA (v) molecules in the gel by acting on the parameters of the

    equation 1 and 2:

    A. Voltage applied

    The voltage determines directly the electric field strength (E) of the Equation 1. But only at low

    voltages the rate of migration of linear DNA fragments is proportional to the voltage applied. At

    high voltages, is also significantly affected and modifies the migration speed. To obtain

    maximum resolution of DNA fragments, the gel should be run at no more than 5V/cm (distance

    between the electrodes).

    B. Agarose concentration

    According to Equation 2, there is a linear relationship between the logarithm of the electrophoretic

    mobility of DNA and the agarose concentration in the gel. Thus, by using gels of different

    concentrations, it is possible to resolve a wide range of DNA molecules (Table 1).

    Table 1: Range of separation in gels containing different amounts of agarose (1 kb = 1 kilobase = 1000 nucleotides)

    Amount of agarose in gel (%[w/v]) Efficient range of separation of linear DNA

    molecules (kb)

    0.3 5-60

    0.6 1-20

    0.7 0.8-10

    0.9 0.5-7

    1.2 0.4-6

    1.5 0.2-3

    2.0 0.1-2

    OH

    O

    O

    O

    OHCH2OH

    O

    OH

    O

    n

    Figure 5: Structure of an agarose gel

    a. Gel

    b. schematic representation of the gel network

    c. basic structure of the monomer

    a b

    c

    -D-galactose 3,6-anhydro--L-galactose

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    Figure 6 Ethidium Bromide structure (up left) and intercalated in DNA (up right). Picture: UV revealing of the ethidium

    bromide bound DNA fragments in an agarose gel.

    C. Molecular size of the DNA

    The electrophoretic mobility of linear double-stranded D