Summary Molecular Tools for Studying GENES AND GENE ACTIVITY
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Molecular Biology
Molecular Tools for Studying Genes and Gene Activity" encapsulates a comprehensive array of techniques and methodologies crucial for unraveling the intricate mechanisms governing genetic expression. This invaluable toolkit empowers researchers to delve into the very essence of life itself, enabling...
Chapter 7 Operon and Chapter 8: Major Shift in Transcription
General Transcription Factors:
RNA Processing II: Capping and Polyadenylation
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TRANSCRIBED BY ; AL-RAISALMILL M. ALAWI
Molecular Tools for Studying GENES AND GENE ACTIVITY ● Instead of a constant current through the gel, this method uses ●
Molecular Separations pulses of current, with relatively long pulses in the forward direction
Gel electrophoresis and shorter pulses in the opposite, or even sideways,direction ●
● It is very often necessary in molecular biology research to separate ● Is valuable for measuring the sizes of DNAs even as large as some
proteins or nucleic acids from each other. of the chromosomes found in yeast.
● For example, we may need to purify a particular enzyme from a Polyacrylamide Gel Electrophoresis ●
crude cellular extract in order to use it or to study its properties. ● Electrophoresis is also often applied to proteins, in which case the
● Gel electrophoresis uses a gel as an anticonvective medium or gel is usually made of polyacrylamide.
sieving medium during electrophoresis, the movement of a charged ● To determine the polypeptide makeup of a complex protein, the
particle in an electric current. experimenter must treat the protein so that the polypeptides, or
●
subunits, will electrophorese independently
●
● This is a horizontal gel made of agarose. The agarose melts at high ● This is usually done by treating the protein with a detergent (sodium
●
temperature, then gels as it cools. dodecyl sulfate, or SDS) to denature the subunits so they no long
● A “comb” is inserted into the molten agarose; after the gel cools, SDS Advantages
the comb is removed, leaving slots, or wells. ● 1. It coats all the polypeptides with negative charges, so they all
● The DNA is then placed in the wells, and an electric current is run electrophorese toward the anode.
through the gel. ● 2. It masks the natural charges of the subunits, so they all
Ion-Ex
● Because the DNA is an acid, it is negatively charged at neutral pH electrophorese according to their molecular masses and not by
●
and electrophoreses, or migrates, toward the positive pole, or their native charges. Small polypeptides fit easily through the pores
anode. in the gel, so they migrate rapidly. Larger polypeptides migrate
●
more slowly
Two-Dimensional Gel Electrophoresis
● The mixture of proteins is electrophoresed through a narrow tube Gel Fi
gel containing molecules called ampholytes that set up a pH ●
gradient from one end of the tube to the other.
● A negatively charged molecule will electrophorese toward the ●
anode until it reaches its isoelectric point, the pH at which it has no
net charge.
● A photograph of a gel after electrophoresis showing the DNA
● Without net charge, it is no longer drawn toward the anode, or the ●
fragments as bright bands.
cathode, for that matter, so it stops.
● DNA binds to a dye that fluoresces orange under ultraviolet light,
● This step is called isoelectric focusing because it focuses proteins
but the bands appear pink in this photograph.
at their isoelectric points in the gel.
Determining the Size of a Large DNA by Gel Electrophoresis ● The gel is removed from the tube and placed at the top of a slab gel Affinit
● 01. The relationship between the log of a DNA’s size and its for ordinary SDS-PAGE. ●
● electrophoretic mobility deviates strongly from linearity if the ● Now the proteins that have been partially resolved by isoelectric
● DNA is very large. focusing are further resolved according to their sizes by ●
● 02. Double-stranded DNA is a relatively rigid rod—very long SDS-PAGE.
and thin. The longer it is, the more fragile it is. In fact, large
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