Sticky and blunt ends

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DNA ends refer to the properties of the end of DNA molecules, which may be sticky ends (cohesive ends), blunt ends or in other forms. The concept is used in molecular biology, especially in cloning or when subcloning inserts DNA into vector DNA. Such ends may be generated by restriction enzymes that cut the DNA – a staggered cut generates two sticky ends, while a straight cut generate blunt ends.

A sticky or cohesive end has protruding single-stranded strands with unpaired nucleotides called overhangs, each overhang can anneal with another complementary one to form base pairs. The two complementary cohesive ends of DNA can anneal together via hydrogen bonding, the stability of these paired ends depends on the melting temperature of the paired overhangs. DNA ligase can join two adjacent strands of DNA by forming a covalent bond between the sugar-phosphate moieties of adjacent nucleotides to join the two together via a phosphodiester bond in a process called ligation. The blunt ends however do not have such protruding strands, and therefore cannot anneal together, and consequently ligation between blunt ends is less efficient.

Single-stranded DNA molecules[edit]

A single-stranded non-circular DNA molecule has two non-identical ends, the 3' end and the 5' end (usually pronounced "three prime end" and "five prime end"). The numbers refer to the numbering of carbon atoms in the deoxyribose, which is a sugar forming an important part of the backbone of the DNA molecule. In the backbone of DNA the 5' carbon of one deoxyribose is linked to the 3' carbon of another by a phosphate group. The 5' carbon of this deoxyribose is again linked to the 3' carbon of the next, and so forth.

Variations in double-stranded molecules[edit]

When a molecule of DNA is double stranded, as DNA usually is, the two strands run in opposite directions. Therefore, one end of the molecule will have the 3' end of strand 1 and the 5' end of strand 2, and vice versa in the other end. However, the fact that the molecule is two stranded allows numerous different variations.

Blunt ends[edit]

The simplest DNA end of a double stranded molecule is called a blunt end. In a blunt-ended molecule both strands terminate in a base pair. Blunt ends are not always desired in biotechnology since when using a DNA ligase to join two molecules into one, the yield is significantly lower with blunt ends. When performing subcloning, it also has the disadvantage of potentially inserting the insert DNA in the opposite orientation desired. On the other hand, blunt ends are always compatible with each other. Here is an example of a small piece of blunt-ended DNA:

5'-CTGATCTGACTGATGCGTATGCTAGT-3'
3'-GACTAGACTGACTACGCATACGATCA-5'

Overhangs and sticky ends[edit]

Non-blunt ends are created by various overhangs. An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. These overhangs are in most cases palindromic.

The simplest case of an overhang is a single nucleotide. This is most often adenosine and is created as a 3' overhang by some DNA polymerases. Most commonly this is used in cloning PCR products created by such an enzyme. The product is joined with a linear DNA molecule with 3' thymine overhangs. Since adenine and thymine form a base pair, this facilitates the joining of the two molecules by a ligase, yielding a circular molecule. Here is an example of an A-overhang:

5'-ATCTGACTA-3'
3'-TAGACTGA-5'

Longer overhangs are called cohesive ends or sticky ends. They are most often created by restriction endonucleases when they cut DNA. Very often they cut the two DNA strands four base pairs from each other, creating a four-base 5' overhang in one molecule and a complementary 5' overhang in the other. These ends are called cohesive since they are easily joined back together by a ligase. Also, since different restriction endonucleases usually create different overhangs, it is possible to cut a piece of DNA with two different enzymes and then join it with another DNA molecule with ends created by the same enzymes. Since the overhangs have to be complementary in order for the ligase to work, the two molecules can only join in one orientation. This is often highly desirable in molecular biology.

For example, these two "sticky" ends are compatible:

5'-ATCTGACT      + GATGCGTATGCT-3'
3'-TAGACTGACTACG        CATACGA-5'

They can form complementary base pairs in the overhang region:

           GATGCGTATGCT-3'
5'-ATCTGACT     CATACGA-5'
3'-TAGACTGACTACG

Frayed ends[edit]

Across from each single strand of DNA, we typically see adenine pair with thymine, and cytosine pair with guanine to form a parallel complementary strand as described below. Two nucleotide sequences which correspond to each other in this manner are referred to as complementary:

5'-ATCTGACT-3'
3'-TAGACTGA-5'
Frayed-dna.png

A frayed end refers to a region of a double stranded (or other multi-stranded) DNA molecule near the end with a significant proportion of non-complementary sequences; that is, a sequence where nucleotides on the adjacent strands do not match up correctly:

5'-ATCTGACTAGGCA-3'
3'-TAGACTGACTACG-5'

The term "frayed" is used because the incorrectly matched nucleotides tend to avoid bonding, thus appearing similar to the strands in a fraying piece of rope.

Although non-complementary sequences are also possible in the middle of double stranded DNA, mismatched regions away from the ends are not referred to as "frayed".

Discovery[edit]

Ronald W. Davis first discovered sticky ends as the product of the action of EcoRI, the restriction endonuclease.[1]

Strength[edit]

Sticky end links are different in their stability. Free energy of formation can be measured to estimate stability. Free energy approximations can be made for different sequences from data related to oligonucleotide UV thermal denaturation curves.[2] Also predictions from molecular dynamics simulations show that some sticky end links are much stronger in stretch than the others.[3]

References[edit]

  1. ^ The Gruber Foundation Homepage | The Gruber Foundation
  2. ^ John SantaLucia Jr. (1997). "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics". Proceedings of the National Academy of Sciences of the USA. 95 (4): 1460–1465. doi:10.1073/pnas.95.4.1460. PMC 19045Freely accessible. PMID 9465037. 
  3. ^ Ehsan Ban and Catalin R Picu (2014). "Strength of DNA Sticky End Links". Biomacromolecules. 15 (1): 143–149. doi:10.1021/bm401425k. PMID 24328228.