#polyacrylamide gel

Sequencing

What is the function of polyacrylamide gel in Sanger sequencing?

In manual Sanger sequencing, termination products from synthesis reactions must be ordered by size to deduce sequence. Denaturing polyacrylamide gel electrophoresis provides single-nucleotide resolution due to its highly crosslinked matrix with small pore size, unlike agarose. Fragments migrate inversely to length; shorter fragments run faster and appear at bottom. By running four lanes or four colors in capillary, the ladder reveals base order. Polyacrylamide does not precipitate DNA, detect bases or primarily buffer pH. Its purpose is size separation with near single-base precision essential

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Electrophoresis

Which matrix is synthesized by polymerizing acrylamide with bisacrylamide?

Polyacrylamide is a synthetic polymer produced by free radical vinyl addition polymerization. Acrylamide monomers polymerize into linear chains, which are crosslinked by N,N'-methylenebisacrylamide to form three-dimensional mesh with defined pore size determined by total monomer percentage and crosslinker ratio. Variation in total concentration and crosslinking tunes resolution for proteins and nucleic acids. Agarose polymerizes by hydrogen bonding upon cooling, not covalent crosslinking. Paper and capillary matrices involve cellulose or silica respectively. The acrylamide-bisacrylamide reacti

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Electrophoresis

What type of support matrix is used in zone electrophoresis?

Zone electrophoresis separates molecules into discrete zones or bands on a stabilizing support to minimize convection and diffusion. Early methods employed filter paper or cellulose acetate strips saturated with buffer, providing a solid anticonvective medium. Samples migrate as discrete zones under applied voltage. Glass and plastic are not porous supports for zone stabilization. Modern zone electrophoresis uses agarose or polyacrylamide gels as improved supports, but historically and categorically, paper represents the classic support matrix distinguishing zone electrophoresis from moving-bo

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Electrophoresis

In polyacrylamide gel formation, what initiates the polymerization reaction in the chemical method?

Chemical polymerization of acrylamide and bisacrylamide requires free radical generation to initiate vinyl addition. Ammonium persulfate (APS) decomposes to produce sulfate free radicals that attack acrylamide double bonds, starting chain propagation. Tetramethylethylenediamine (TEMED) acts as a catalyst by accelerating APS decomposition and facilitating radical formation at room temperature, stabilizing the reaction and controlling gelation rate. Neither acts efficiently alone for rapid casting. Riboflavin initiates photochemical polymerization under UV light, not chemical initiation. Glycero

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Electrophoresis

Which of the following gel matrices is best suited for resolving DNA fragments ranging from 50 to 50,000 bp?

Agarose forms a porous matrix with large, tunable pore sizes determined by concentration, typically 0.7 to 2 percent. This range allows efficient sieving of DNA fragments from approximately 50 bp to over 50,000 bp, covering routine plasmids, restriction digests, and genomic DNA preparations. Polyacrylamide offers much smaller pores, ideal for 5 to 500 bp resolution at single-basepair discrimination. Cellulose acetate and thin-layer matrices lack molecular sieving for nucleic acids. Agarose provides low electroendosmosis, low UV absorption, and non-denaturing separation, making it the standard

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Electrophoresis

Which gel percentage has the largest pore size?

Polyacrylamide gel pore diameter is inversely proportional to total acrylamide concentration and crosslinker percentage, because higher monomer density creates denser polymer network. Lower percentage yields fewer chains and larger interstitial spaces, permitting relatively unhindered migration of high-molecular-weight proteins. Five percent gel offers very open matrix suitable for resolving proteins above 200 kilodaltons, whereas 10 percent, 12 percent and 15 percent produce progressively tighter networks for medium to low molecular weight separation. Therefore, among given options, 5 percent

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Electrophoresis

What is the function of TEMED in gel preparation?

Tetramethylethylenediamine accelerates polyacrylamide gel formation by catalyzing decomposition of ammonium persulfate into reactive sulfate free radicals and stabilizing their activity through electron donation. Without TEMED, radical generation proceeds slowly, leading to incomplete or excessively delayed polymerization and poor pore reproducibility. Acrylamide plus bisacrylamide crosslinking forms porous matrix essential for molecular sieving during electrophoresis. Visualization functions belong to Coomassie, silver or fluorescent dyes, while DNA stains like ethidium bromide intercalate nu

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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