DNA end
Encyclopedia
DNA end or sticky end refers to the properties of the end of a molecule
of DNA
or a recombinant DNA molecule. The concept is important in molecular biology
, especially in cloning or when subcloning inserts DNA into vector DNA. All the terms can also be used in reference to RNA
. The sticky ends or cohesive ends form base pairs. Any two complementary cohesive ends can anneal, even those from two different organisms. This bondage is temporary however, and DNA ligase will eventually form a covalent bond between the sugar-phosphate residue of adjacent nucleotides to join the two molecules together.
(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.
. 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'
s 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 polymerase
s. 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
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'
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".
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or a recombinant DNA molecule. The concept is important in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, especially in cloning or when subcloning inserts DNA into vector DNA. All the terms can also be used in reference to RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
. The sticky ends or cohesive ends form base pairs. Any two complementary cohesive ends can anneal, even those from two different organisms. This bondage is temporary however, and DNA ligase will eventually form a covalent bond between the sugar-phosphate residue of adjacent nucleotides to join the two molecules together.
Single-stranded DNA molecules
A single-stranded non-circular DNA molecule has two non-identical ends, the 3' end and the 5' endDirectionality (molecular biology)
Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5′-end and a 3′-end...
(usually pronounced "three prime end" and "five prime end"). The numbers refer to the numbering of carbon atoms in the deoxyribose
Deoxyribose
Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...
, 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
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
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
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
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 pairBase pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
. Blunt ends are not always desired in biotechnology since when using a DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
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
Non-blunt ends are created by various overhangs. An overhang is a stretch of unpaired nucleotideNucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s 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 polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
s. 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
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
overhangs. Since adenine and thymine form a base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called 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
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
.
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
Across from each single strand of DNA, we typically see adenineAdenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
pair with thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
, and cytosine pair with guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
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'
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".