#HindIII

Restriction Digestion & Mapping

Match: HindIII → ?

HindIII is a classic type II restriction enzyme widely used in NCERT textbook cloning illustrations and commercial vectors. It recognizes palindromic hexamer 5'-AAGCTT-3' and cuts between the two adenines on each strand, leaving four-base 5' overhangs AGCT that are cohesive and easily ligated. Recognizing sequence-specificity allows prediction of sticky end complementarity, calculation of fragment numbers after digestion, and design of compatible vectors like pBR322 which contains single HindIII site in tetracycline gene. Memorizing common palindromes including EcoRI GAATTC and HindIII AAGCTT

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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Restriction Digestion & Mapping

How many HindIII sites are present if digestion gives 1200 and 3800 bp bands?

Total size of a circular plasmid is sum of all fragments produced by complete restriction digestion, visualized by agarose electrophoresis. Observing two bands of 1200 bp and 3800 bp gives total 5000 bp, indicating full digestion into two fragments without loss. In circular DNA, number of fragments equals number of recognition sites cut, assuming enzyme cuts to completion. One site would linearize plasmid into single 5000 bp band. Two distinct bands therefore imply two HindIII sites spaced 1200 bp apart on one arc and 3800 bp on the other arc, confirming presence of second site for mapping.

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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Restriction Digestion & Mapping

Which fragment profile confirms correct orientation in HindIII+PstI screening?

Orientation screening using two enzymes, one cutting in vector backbone and one asymmetrically within insert, generates orientation-specific junction fragments whose sizes differ with insert direction. Correct orientation places internal sites at predicted separation producing characteristic larger and smaller bands calculated from map, while reverse orientation alters distance between sites yielding different fragment sizes. Clone C exhibits size combination consistent with forward transcription direction, intact reading frame, and expected restriction map summed from plasmid and insert seque

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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Restriction Digestion & Mapping

Which clone orientation produces 1.7 kb and 4.1 kb fragments on EcoRI+HindIII digestion?

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.

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Restriction Digestion & Mapping

What type of fragment is obtained from EcoRI+HindIII if both create sticky ends?

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.

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