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#sticky ends

10 public questions tagged with this topic.

ZOOLOGY

Which enzyme is used to produce sticky ends in a DNA fragment?

Restriction endonucleases cut DNA at specific sequences, generating sticky ends for recombination. This follows from latest NCERT 2026-27 principle explaining the concept clearly for NEET students in simple steps as per rationalized syllabus.

Ref: NCERT Biology Textbook - Latest Edition for Academic Session 2026-27 (Zoology section, Rationalized Textbook for Class XI and XII), Chapter: Biology - Zoology portion (Latest NCERT Textbooks for Academic Session 2026-27 - Rationalized Edition for Class XI and XII), Topic: Structural organization, physiology, human health and related concepts as per latest syllabus.

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RDT Basics

Which type of cloning uses short synthetic oligonucleotides with sticky ends?

Adaptor ligation uses short synthetic duplex oligonucleotides of ten to twenty base pairs engineered to carry compatible sticky ends without requiring internal cleavage. Adaptors are formed by annealing two partially complementary oligonucleotides, generating a double-stranded region with one cohesive extension that matches ends created by restriction enzymes. When incubated with target fragments and T4 DNA ligase, adaptors covalently attach, modifying termini for subsequent insertion into vectors. This approach surpasses blunt-end cloning in efficiency, avoids exonuclease trimming, and is fundamental for next-generation sequencing library preparation, cDNA cloning, and addition of defined ends.

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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RDT Basics

Enzyme that produces sticky ends at C/TCGAG:

XhoI, derived from Xanthomonas holcicola, is a Type II restriction enzyme recognizing hexameric palindrome 5'-C^TCGAG-3' and cutting after first base to leave four-base 5' overhang TCGA. Cleavage requires magnesium and produces cohesive ends compatible with SalI, compatible with XhoI-generated fragments, and useful for creating complementary junctions. The enzyme cuts rarely in AT-rich genomes due to GC-rich recognition, making it attractive for constructing expression cassettes, removing promoters, and performing directional cloning where unique sites are needed to maintain reading frame and transcription orientation.

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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RDT Basics

Which strategy converts blunt ends to sticky ends?

Homopolymer tailing overcomes incompatibility of blunt ends by enzymatically synthesizing complementary extensions. Terminal deoxynucleotidyl transferase adds homopolymer tracts such as poly-deoxyguanosine to vector 3' ends and poly-deoxycytosine to insert ends. Guanine-cytosine pairing provides high thermal stability due to three hydrogen bonds, enabling annealed complexes to be extended or ligated and subsequently repaired in vivo. Alternative dA-dT tailing is also used. Before advent of synthetic linkers, this method was central to first cDNA cloning experiments, demonstrating conversion of blunt molecules into clonable sticky structures without restriction site dependence.

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 Enzyme and Vector

Sticky ends are most efficient in ligation because:

Sticky ends are produced when restriction enzymes cut asymmetrically within recognition sites, leaving short single-stranded overhangs. Ligation efficiency depends on transient association of fragments before phosphodiester bond sealing by DNA ligase. Cohesive termini can anneal through Watson-Crick hydrogen bonding between complementary bases, forming a stable, pre-aligned substrate that greatly increases effective local concentration of 5' phosphate and 3' hydroxyl groups. Blunt ends lack this stabilization and rely on random collision. Complementary pairing therefore lowers activation energy, improves fidelity and accelerates recombinant formation in cloning.

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 enzyme is used for labeling sticky ends?

Labeling cohesive ends commonly employs DNA polymerases that can fill in 5' overhangs. T4 DNA polymerase possesses strong 3' to 5' exonuclease and polymerase activity that efficiently incorporates labeled dNTPs opposite the single-stranded overhang using the protruding 5' sequence as template. EcoRI is restriction enzyme that creates ends, not label them. Ligase joins fragments, exonuclease III degrades from 3' terminus. Hence T4 polymerase is standard choice for labeling sticky ends in mapping, footprinting, and terminal labeling protocols using radioactive or fluorescent nucleotides.

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 enzyme creates sticky ends?

EcoRI recognizes GAATTC and creates a staggered cut between G and A on both strands, leaving AATT 5' overhangs that are single-stranded and complementary, termed sticky or cohesive ends. DNase I is a nonspecific endonuclease that degrades DNA randomly. Taq and Pfu polymerases are thermostable DNA synthesizers used in PCR, not restriction enzymes, and they add non-templated A or produce blunt products respectively. Therefore only EcoRI among listed creates characteristic cohesive termini useful for directional cloning and efficient ligation via complementary base pairing.

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

Sticky vs blunt ends are distinguished by:

Distinguishing cohesive versus blunt termini relies on substrate specificity of labeling enzymes. DNA polymerase I Klenow fragment and T4 DNA polymerase require a single-stranded template to incorporate labeled dNTPs, a condition fulfilled by 5' overhanging sticky ends with recessed 3'-OH. Blunt ends lack overhangs for templated fill-in. Therefore only sticky-ended fragments become radioactive upon incubation with labeled nucleotides, while blunt fragments stay unlabeled. Agarose pattern shows total DNA, while western blot and RT-PCR detect proteins and RNA, not relevant for terminus typing.

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 similarly double-digested vector backbone during recombinant construction.

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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