How can a restriction enzyme leave sticky ends between DNA fragments?

Answer 1

The sticky ends generated by restriction enzymes can easily be joined using an enzyme called ligase. Blunt ends however, cannot be joined so easily. This is why restiction enzymes that create sticky ends are more useful.

If blunt ends result, small segments called modifiers are attached to the sticky ends. These modifiers are nucleotide sequences that have sticky ends and attach to the blunt ends, thus making them sticky ends.

Answer 2

Restriction enzymes create staggered cuts in the DNA sequence, resulting in overhanging or sticky ends. These ends have unpaired bases which can easily anneal to complementary sequences in another DNA fragment, facilitating their joining through base pairing and DNA ligase activity. This enables the creation of recombinant DNA molecules.

Answer 3

DNA cut with restriction enzymes that produce 'sticky ends' can be annealed with DNA from another source cut by the same enzyme. The ends complement one another according to the nucleotide pairing code and then the enzyme DNA ligase is used to reseal the sugar phosphate backbones.

.....CACAGT + AG...... annealed makes ......CACACT-AG..... and then DNA ligase fills

.....GT GTCATC...... ......GT-GTGATC......

the gaps represented by hyphens.

If a restriction enzyme yields blunt ends such as

.....CACAT cannot use the overlaps in sticky ends to exploit the nucleotide pairing code

.....GTGTA

but nevertheless special techniques and kits can be used to achieve blunt end recombination where the second DNA must be cut with the same or another blunt end cutter. The methods are much less efficient but they are doable.

Brian Dancer, Caerleon, S.Wales UK DNA cut with restriction enzymes that produce 'sticky ends' can be annealed with DNA from another source cut by the same enzyme. The ends complement one another according to the nucleotide pairing code and then the enzyme DNA ligase is used to reseal the sugar phosphate backbones.

.....CACAGT + AG...... annealed makes ......CACACT-AG..... and then DNA ligase fills

.....GT GTCATC...... ......GT-GTGATC......

the gaps represented by hyphens.

If a restriction enzyme yields blunt ends such as

.....CACAT cannot use the overlaps in sticky ends to exploit the nucleotide pairing code

.....GTGTA

but nevertheless special techniques and kits can be used to achieve blunt end recombination where the second DNA must be cut with the same or another blunt end cutter. The methods are much less efficient but they are doable.

Brian Dancer, Caerleon, S.Wales UK

Answer 4

A portion of the Answer appears in the Q'n [i. e. 'specific']. The Answer is: utter Specificity. Restriction enzymes may bind, and thereafter act, only at Specific 'Recognition Sequences'; of which there are millions to 'choose' from.

Answer 5

They can - and are - it's just harder. Non-sticky ends (typically called blunt ends) are routinely used in making recombinant DNA, particularly when there's no easily available cutting sites for sticky-ended restriction enzymes. The advantage of sticky ends is that they are specific (i.e. a sticky end cut by one enzyme will only ligate with a sticky end cut by another) and because the hydrogen bonding between bases of the sticky ends hold the ends together such that there is frequently a substrate available to be ligated by DNA ligase. Blunt ends lack this specificity and can be ligated onto any other blunt end and because there are no hydrogen bonding to hold blunt end joins together, the rate of the ligation reaction is far more inefficient.

That said, there are now many techniques that do not call for restriction enzymes at all to create recombinant DNA. Many of these techniques, which use recombinases and transposases to insert DNA molecules with the proper flanking sequences, in fact require blunt ended DNA to work properly.

Answer 6

DNA Ligase