8 public questions tagged with this topic.
Gel electrophoresis separates DNA fragments based on size, allowing for further analysis or cloning.
Ref: NCERT Class 12 Biology Chapter 9: Biotechnology: Principles and Processes Processes - Isolation of DNA and Gel Electrophoresis
Determining insert orientation in a plasmid requires direction-sensitive detection. Amplification using one primer annealing within the insert and another annealing in the vector backbone produces a product only when the insert is in a defined orientation and yields size indicating forward or reverse cloning. Using two insert-specific primers merely confirms insert presence, independent of vector context. Single-site restriction digestion linearizes plasmid regardless of orientation, while two vector sites excise insert but do not distinguish its direction. This vector-insert PCR strategy is q
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.
Amplified fragment length polymorphism combines advantages of restriction digestion and PCR amplification. Genomic DNA is first cut with two restriction enzymes, typically EcoRI and MseI, and adapters are ligated to sticky ends. Subsequent pre-selective and selective PCR using adapter-complementary primers with additional nucleotides amplifies a subset of fragments. This generates highly reproducible, multilocus fingerprints without prior sequence knowledge. RAPD uses only random PCR, SSR uses only locus-specific PCR, and SNP genotyping detects single base variations, lacking this dual enzymat
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.
Agarose gel analysis of recombinant plasmid digested with enzymes flanking insertion site normally separates vector backbone from insert as two bands. If vector alone is 3 kb and after cloning digestion still shows only a single 3 kb band of double intensity, interpretation requires understanding overlapping migration. Total recombinant is 6 kb; digestion releases 3 kb vector and 3 kb insert of identical size that co-migrate as one intense band. Without additional enzymes or size markers, single band may be misinterpreted, but careful intensity comparison indicates insert size equals vector si
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.
When linear DNA is cut by two enzymes producing three fragments, each fragment has two ends derived from cleavage events. Ends originating from natural termini or restriction cuts may be blunt or sticky depending on enzyme specificity. If only 2 kb fragment becomes labeled after end-filling with Klenow and radiolabeled dNTPs, it must be the only fragment possessing a 5' overhang with recessed 3' OH that polymerase can extend using template strand. Other fragments bearing blunt ends or 3' overhangs cannot incorporate label efficiently, thus selective labeling marks fragments with appropriate co
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.
Insert orientation determines relative positions of internal restriction sites to vector backbone sites. In one orientation, EcoRI and HindIII may be close together, producing one very small fragment that may be overlooked and one almost full-length band resembling empty vector. Flipping insert swaps distances, placing EcoRI site distant from HindIII site, generating 1.7 kb and 4.1 kb fragments that sum to total recombinant size of 5.8 kb. Observing these distinct sizes allows unambiguous discrimination between forward and reverse clones, critical for ensuring correct promoter-driven expressio
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.
When a heteroduplex containing a mismatched restriction site undergoes mismatch repair, outcome depends on repair efficiency and strand bias. With complete repair, all molecules regain the restriction site and show uniform digestion pattern after electrophoresis. With no repair, all retain mismatch and show parental uncut pattern. At fifty percent efficiency, population becomes heterogeneous: half molecules are repaired and cleavable, half remain resistant. Gel then displays both parental and repaired bands simultaneously, reflecting mixed population important for interpreting heteroduplex ana
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.
EcoRI recognizes GAATTC and HindIII recognizes AAGCTT, both cutting asymmetrically within the palindromic site to leave four-nucleotide 5' overhangs that are cohesive or sticky and complementary to themselves. When a DNA fragment is excised using these two different sticky-end cutters, each terminus retains its characteristic single-stranded extension derived from its own site. Self-ligation is prevented because ends are incompatible. Therefore the isolated insert carries two different sticky ends, one EcoRI-derived and one HindIII-derived, enabling efficient directional insertion into a simil
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.
