There are 8 common varieties of There are 8 common varieties of nucleotides, each consists of 3 part:nucleotides, each consists of 3 part:

A nitrogen based A nitrogen based A pentose sugar, A pentose sugar, either deoxyribose or riboseeither deoxyribose or ribose At least one phosphate groupAt least one phosphate group

NH

NN

N

purine

N

N

pyrimidine

NH

ONH

O

thymine

N

ONH

H2N

cytosine

NH

ONH

O

uracil

Cytosine (C), thymine (T) and Uracil (U) have single rings and they are pyrimidinine

NITROGEN BASEDNucleotides can divided into 2 class or groupNucleotides can divided into 2 class or group

adenine guanine

Adenine (A) and guanine (G) are double-ringed molecule of class called purine

N

N

NH2

N

NH

HN

NNH2N

HN

O

Purine form bonds to a five-carbon Purine form bonds to a five-carbon sugar (a pentose) via their N9 atomssugar (a pentose) via their N9 atoms

Pyrimidines do so through their N1 Pyrimidines do so through their N1 atomsatoms

PENTOSE SUGARPENTOSE SUGAR

In ribonucleotides, the In ribonucleotides, the pentose is ribosepentose is ribose

In deoxyribonucleotide (or In deoxyribonucleotide (or deoxynucleotides) the deoxynucleotides) the sugar is 2’-deoxyribose – sugar is 2’-deoxyribose – the carbon at position 2’ the carbon at position 2’ lacks a hydroxyl grouplacks a hydroxyl group

In ribononucleotide, one or more phosphate groups is In ribononucleotide, one or more phosphate groups is bonded to atom C3’ or atom C5’ of the pentose to form a bonded to atom C3’ or atom C5’ of the pentose to form a 3’-nucleotide or a 5’-nucleotide.3’-nucleotide or a 5’-nucleotide.

When phosphate group is absent, the compound is When phosphate group is absent, the compound is known nucleoside.known nucleoside.

Ribonucleotides are found in RNA (ribonucleic acid)Ribonucleotides are found in RNA (ribonucleic acid)

Deoxynucleotides are found in DNA (deoxyribonucleic Deoxynucleotides are found in DNA (deoxyribonucleic acid)acid)

A, G, C found as both RNA and DNAA, G, C found as both RNA and DNA U is found as RNAU is found as RNA T is found as DNAT is found as DNA

DefinitionsDefinitions

DNA stands for deoxyribonucleic acid. It is DNA stands for deoxyribonucleic acid. It is the genetic code molecule for most the genetic code molecule for most organisms.organisms.

RNA stands for ribonucleic acid. RNA RNA stands for ribonucleic acid. RNA molecules are involved in converting the molecules are involved in converting the genetic information in DNA into proteins. genetic information in DNA into proteins. In retroviruses, RNA is the genetic In retroviruses, RNA is the genetic material.material.

Nucleotide and Nucleotide Nucleotide and Nucleotide derivativesderivatives

The bulk of nucleotides in any cell are found in The bulk of nucleotides in any cell are found in polymeric form, as either DNA and RNApolymeric form, as either DNA and RNA

Primary function are information storage and Primary function are information storage and transfertransfer

Free nucleotides and nucleotides derivatives Free nucleotides and nucleotides derivatives perform an enormous variety of metabolic perform an enormous variety of metabolic functions and not related to management of functions and not related to management of genetic informationgenetic information

The best known is ATP (adenosine triphosphate) contains adenine, The best known is ATP (adenosine triphosphate) contains adenine, ribose and triphosphate groupribose and triphosphate group

ATP is the principal, short –term, recyclable energy supply for cells. ATP is the principal, short –term, recyclable energy supply for cells. When the phostphate bonds of ATP are broken, a significant amount When the phostphate bonds of ATP are broken, a significant amount of energy is release.of energy is release.

More energy is release from phosphate bonds than is release from More energy is release from phosphate bonds than is release from most other covalent bonds. For this reason, the phosphate-phosphate most other covalent bonds. For this reason, the phosphate-phosphate bonds of ATP are known as high-energy bondsbonds of ATP are known as high-energy bonds

Energy is release when ATP is convert to ADP, and when phosphate Energy is release when ATP is convert to ADP, and when phosphate is remove from ADP to form AMP, though the latter reaction is not as is remove from ADP to form AMP, though the latter reaction is not as common in cellscommon in cells

Energy release from the phosphate bonds of ATP is used for Energy release from the phosphate bonds of ATP is used for important life-sustaining activities, such as synthesis reactions, important life-sustaining activities, such as synthesis reactions, locomotion, and transportation of substance into and out of cells.locomotion, and transportation of substance into and out of cells.

Cells also use ATP as a structural molecule in formation of Cells also use ATP as a structural molecule in formation of coenzymes.coenzymes.

Nucleic acid structureNucleic acid structure

Segment of DNA ChainSegment of DNA Chain

NCCH

C

N

NC

C

N

O

NH2-2

O3POO

CH2

H

O

H

H

HH

NC CH

N

O

CC

O

CH3

O P

O

OO

CH2

H

O

H

H

HH

NC CH

N

O

CHC

NH2

O P

O

OO

CH2

H

OH

H

H

HH

5’-end

3’-end

guanine

thymine

cytosine3’-5’link

Nucleic acid structureNucleic acid structure Nucleotides can be joined to each other to form RNA and DNA. Nucleotides can be joined to each other to form RNA and DNA.

The nucleic acids are chains of nucleotides whose phosphates bridge the 3' and 5' The nucleic acids are chains of nucleotides whose phosphates bridge the 3' and 5' positions of neighboring ribose units positions of neighboring ribose units

The phosphates of these The phosphates of these polynucleotides polynucleotides are acidic, so at physiological pH, nucleic are acidic, so at physiological pH, nucleic acids are polyanions. acids are polyanions.

The linkage between individual nucleotides is known as a The linkage between individual nucleotides is known as a phosphodiester bondphosphodiester bond. .

Each nucleotide that has been incorporated into the polynucleotide is known as a Each nucleotide that has been incorporated into the polynucleotide is known as a nucleotide residue. nucleotide residue.

The terminal residue whose C5' is not linked to another nucleotide is called the The terminal residue whose C5' is not linked to another nucleotide is called the 5' end5' end

The terminal residue whose C3' is not linked to another nucleotide is called the 3' The terminal residue whose C3' is not linked to another nucleotide is called the 3' end. end.

By convention, the sequence of nucleotide residues in a nucleic acid is written, left to By convention, the sequence of nucleotide residues in a nucleic acid is written, left to right, from the 5' end to the 3' end. right, from the 5' end to the 3' end.

Or: pdGpdTpdC

Or: pd(GTC)RNA abbreviations lack the d (for deoxy)

PP P

G T CHO

5'

'3d d d

DNA and RNA chains are abbreviated DNA and RNA chains are abbreviated using a structure where vertical lines using a structure where vertical lines represent the sugars, diagonal lines with P represent the sugars, diagonal lines with P at the midpoint represent the 3,5-at the midpoint represent the 3,5-phosphodiester bonds, and horizontal phosphodiester bonds, and horizontal lines the ends of the chain.lines the ends of the chain.

The structures are always written with the The structures are always written with the 5’ end to the left.5’ end to the left.

Single letter abbreviations are also used.Single letter abbreviations are also used.

The base composition of DNAThe base composition of DNA

Though there appear to be no rules governing the Though there appear to be no rules governing the nucleotide composition of typical RNA molecules, nucleotide composition of typical RNA molecules,

DNA has equal numbers of adenine and thymine DNA has equal numbers of adenine and thymine residues (A = T) and equal numbers of guanine and residues (A = T) and equal numbers of guanine and cytosine (G = C). cytosine (G = C).

These relationships, known as These relationships, known as Chargaff’s rules. Chargaff’s rules.

The significance of Chargaff's rules was not immediately The significance of Chargaff's rules was not immediately appreciated, but we now know that the structural is for appreciated, but we now know that the structural is for the rules derives from DNA's double-stranded nature.the rules derives from DNA's double-stranded nature.

The double helixThe double helixWatson-Crick structure:Watson-Crick structure:

Two polynucleotide chains wind Two polynucleotide chains wind around a common axis to form a around a common axis to form a double double helixhelix. .

The two strands of DNA are The two strands of DNA are antiparallelantiparallel, but each forms a right-, but each forms a right-handed helix. handed helix.

The bases occupy the core of the helix The bases occupy the core of the helix and sugar-phosphate chains run along and sugar-phosphate chains run along the periphery, thereby minimizing the the periphery, thereby minimizing the repulsions between charged repulsions between charged phosphate groups. phosphate groups.

The surface of the double helix The surface of the double helix contains two grooves of unequal width: contains two grooves of unequal width: the the major major and and minor grooves. minor grooves.

The double helixThe double helix Each base is hydrogen bonded Each base is hydrogen bonded

to a base in the opposite strand to a base in the opposite strand to form a planar to form a planar base pair. base pair.

Each adenine residue must pair Each adenine residue must pair with a thymine residue and vice with a thymine residue and vice versa, and each guanine versa, and each guanine residue must pair with a residue must pair with a cytosine residue and vice versa.cytosine residue and vice versa.

These hydrogen bonding These hydrogen bonding interactions, a phenomenon interactions, a phenomenon known as known as complementary complementary base pairingbase pairing, , result in the result in the specific association of the two specific association of the two chains of the double helix. chains of the double helix.

Single stranded nucleic acidSingle stranded nucleic acid Single-stranded DNA is rareSingle-stranded DNA is rare

In In contrast, RNA occurs primarily as single strands, which usually contrast, RNA occurs primarily as single strands, which usually form compact structures rather than loose extended chains form compact structures rather than loose extended chains

A RNA strand-which is identical to a DNA strand except for the A RNA strand-which is identical to a DNA strand except for the presence of OH groups and the substitution of uracil for thymine-can presence of OH groups and the substitution of uracil for thymine-can base-pair with a complementary strand of RNA or DNA. base-pair with a complementary strand of RNA or DNA.

As expected, A pairs with U or T in DNA, and G with C.As expected, A pairs with U or T in DNA, and G with C.

Base pairing often occurs intramoleculy giving rise to stem-loop Base pairing often occurs intramoleculy giving rise to stem-loop structures or, when loops interact with each other, to more complex structures or, when loops interact with each other, to more complex structures. structures.

The DNA helixThe DNA helixThe geometry of DNAThe geometry of DNA

The biologically most common form of DNA is known as B-DNA, - The biologically most common form of DNA is known as B-DNA, - structural features first noted by Watson and Crick together with structural features first noted by Watson and Crick together with Rosalind Franklin and other. Rosalind Franklin and other.

Double-helical DNA can assume several distinct structural Double-helical DNA can assume several distinct structural depending on the solvent composition and base sequence. The depending on the solvent composition and base sequence. The major structural variants of DNA are A- and Z-DNA. major structural variants of DNA are A- and Z-DNA.

Under dehydrating conditions, B-DNA undergoes a reversible Under dehydrating conditions, B-DNA undergoes a reversible conformational change to A-DNA which forms a wider and flatter conformational change to A-DNA which forms a wider and flatter right-handed helix than does B-DNA. right-handed helix than does B-DNA.

Key to structure. Structural features of ideal A-, B- and Z-DNA.Key to structure. Structural features of ideal A-, B- and Z-DNA.

B DNA segmentB DNA segment

Sugar-phosphate backbone

Hydrogen bondedbase pairs in thecore of the helix

Chain 1

Chain 2

B DNA: 2B DNA: 2

Outside diameter, 2 nm

Length of one turn of helix is 3.4 nm and contains 10 base pairs.

Interior diameter, 1.1 nm

Major groove

Minor groove

A DNA and Z DNAA DNA and Z DNA

A second form of DNA is the A form.A second form of DNA is the A form. It has 11 base pairs per turn of the helix It has 11 base pairs per turn of the helix

and the bases lie at an angle of about 20and the bases lie at an angle of about 20oo relative to the helix axis. It, too, is a right relative to the helix axis. It, too, is a right hand double helix.hand double helix.

A third form of DNA is the Z form. It is a A third form of DNA is the Z form. It is a left handedleft handed helix. helix.

A picture of A DNA is on the next slide.A picture of A DNA is on the next slide.

A DNA segmentA DNA segment

Base pairs notperpendicular tohelix axis. 11 pairsper turn.

Force stabilizing nucleic acid Force stabilizing nucleic acid structure - DNAstructure - DNA

Denaturation and renaturationDenaturation and renaturation

When a solution of duplex DNA is heated above When a solution of duplex DNA is heated above a characteristic temperature, its native structure a characteristic temperature, its native structure collapses and its two complementary strands collapses and its two complementary strands separate and assume randam conformations. separate and assume randam conformations.

The stability of the DNA double helix, and its TThe stability of the DNA double helix, and its Tmm (melting temperature) depends on several (melting temperature) depends on several factors including factors including

- the nature of the solvent, - the nature of the solvent, - the identities and concentration of the - the identities and concentration of the

ions in solution, ions in solution, -and the pH. -and the pH.

TTmm also increases linearly with the mole fraction also increases linearly with the mole fraction of G.C base pair, although this is not, as one of G.C base pair, although this is not, as one might expect, entirely because G.C base pairs might expect, entirely because G.C base pairs contain one more hydrogen bond than A.T base contain one more hydrogen bond than A.T base pair.pair.

Base pairingBase pairing Base pairing is apparently a “glue” that holds together double-Base pairing is apparently a “glue” that holds together double-

stranded nucleic acids. stranded nucleic acids.

Only Watson-crick pairs occur in the crystal structure of self-Only Watson-crick pairs occur in the crystal structure of self-complementary oligonucleotides. Other hydrogen-bonded base complementary oligonucleotides. Other hydrogen-bonded base pairs with reasonable geometric are known.pairs with reasonable geometric are known.

Observation indicate that Watson-crick geometry is the most stable Observation indicate that Watson-crick geometry is the most stable mode of base pairing in the double helix, even though non Watson-mode of base pairing in the double helix, even though non Watson-crick base pairs are theorically possible. crick base pairs are theorically possible.

Hydrogen bonding contribute little to the stability of nucleic acid Hydrogen bonding contribute little to the stability of nucleic acid structure. This is because, on denaturation, the hydrogen bonds structure. This is because, on denaturation, the hydrogen bonds between the base pair of native nucleic acid are replaced by between the base pair of native nucleic acid are replaced by energetically similar hydrogen bonds between the bases and water. energetically similar hydrogen bonds between the bases and water. Only types of forces must therefore play an important role in Only types of forces must therefore play an important role in stabilizing nucleic acid structure.stabilizing nucleic acid structure.

Base stacking and hydrophobic Base stacking and hydrophobic interaction interaction

Stacking interactions are a form of van der waals Stacking interactions are a form of van der waals interaction. interaction.

Interaction between stacked G and C bases are greater Interaction between stacked G and C bases are greater than those between stacked A and T bases, which than those between stacked A and T bases, which largely accounts for the greater thermal stability of DNAs largely accounts for the greater thermal stability of DNAs with a high G+C content. with a high G+C content.

Note also that differing sets of base pairs in a stack have Note also that differing sets of base pairs in a stack have different staking energies. Thus, the stacking energy of a different staking energies. Thus, the stacking energy of a double helix is sequence-dependent.double helix is sequence-dependent.

RNARNA

RNA is stabilizing by the same force that RNA is stabilizing by the same force that stabilizing DNA. stabilizing DNA.

In fact RNA may be even more rigid than In fact RNA may be even more rigid than DNA to presence of greater number of water DNA to presence of greater number of water molecule that form hydrogen bond to the 2’-molecule that form hydrogen bond to the 2’-OH group of DNA.OH group of DNA.

RNAs may contain double-stranded segmentsRNAs may contain double-stranded segments

Fractionation of nucleic acidsFractionation of nucleic acidsMost of common procedures for isolating and Most of common procedures for isolating and

characterizing protein, often with some modification, characterizing protein, often with some modification, are also used to fractionate nucleic acid according to are also used to fractionate nucleic acid according to size, composition and sequence. Some of the most size, composition and sequence. Some of the most useful nucleic acid fractionation procedure areuseful nucleic acid fractionation procedure are

ChromatographyChromatography ElectrophoresisElectrophoresis UltracentrifugationUltracentrifugation

Chromatography Chromatography Many of the chromatographic techniques that are used to separate proteins Many of the chromatographic techniques that are used to separate proteins

also apply to nucleic acids. also apply to nucleic acids.

Hydroyapatite, a form of calcium phosphate is particularly useful in the Hydroyapatite, a form of calcium phosphate is particularly useful in the chromatographic purification and fractionation of DNA. chromatographic purification and fractionation of DNA.

Double stranded DNA binds to hydroyapatite more tightly than do most Double stranded DNA binds to hydroyapatite more tightly than do most other molecules. DNA can be rapidly isolated by passing a cell lysate other molecules. DNA can be rapidly isolated by passing a cell lysate through a hydroyapatite column, washing the column with a low through a hydroyapatite column, washing the column with a low concentration of phosphate buffer to release only the RNA and protein, and concentration of phosphate buffer to release only the RNA and protein, and then eluting the DNA with a concentrated phosphate solution. then eluting the DNA with a concentrated phosphate solution.

Affinity chromatography is used to isolate specific nucleic acids. Affinity chromatography is used to isolate specific nucleic acids.

For example, most eukaryotic messenger RNAs (mRNAs) have a poly (A) For example, most eukaryotic messenger RNAs (mRNAs) have a poly (A) sequences or cellulose to which poly (U) is covalently attached. The sequences or cellulose to which poly (U) is covalently attached. The poly(A) sequences specifically bind to the complementary poly(U) in high poly(A) sequences specifically bind to the complementary poly(U) in high salt and low temperature and can later be released by altering these salt and low temperature and can later be released by altering these condition.condition.

Electrophoresis Electrophoresis Gel electrophoresis Gel electrophoresis is a technique for is a technique for

separating molecules (including fragments of separating molecules (including fragments of nucleic acids) by size, shape, and electrical nucleic acids) by size, shape, and electrical charge. charge.

The technique involves drawing DNA The technique involves drawing DNA molecules, which have an overall negative molecules, which have an overall negative charge, through a semisolid gel by an electric charge, through a semisolid gel by an electric current toward the positive electrode within an current toward the positive electrode within an electrophoresis chamber.electrophoresis chamber.

The gel is typically composed of a purified The gel is typically composed of a purified sugar component of agar called agarose. sugar component of agar called agarose.

Smaller DNA fragments move faster and farther Smaller DNA fragments move faster and farther than larger ones. Saiz of a fragments detrmined than larger ones. Saiz of a fragments detrmined by comparing the distance it travels to the by comparing the distance it travels to the distances traveled by standard DNA fragment of distances traveled by standard DNA fragment of known size. known size.

In genetic engineering, scientists use the In genetic engineering, scientists use the technique to isolate fragments of DNA technique to isolate fragments of DNA molecules that can then be inserted into vectors, molecules that can then be inserted into vectors, multiplied by PCR, or preserved in a gene multiplied by PCR, or preserved in a gene library. library.

Southern blotSouthern blot The The Southern blot Southern blot technique begins with the procedures of gel electrophoresis just technique begins with the procedures of gel electrophoresis just

described, but allows researchers to stabilize specific DNA sequences and then described, but allows researchers to stabilize specific DNA sequences and then localize them using DNA dyes or probes. localize them using DNA dyes or probes.

Once the DNA fragments have been separated by size, the liquid in the Once the DNA fragments have been separated by size, the liquid in the electrophoresis gel is blotted out, the DNA is denatured with NaOH, and its single electrophoresis gel is blotted out, the DNA is denatured with NaOH, and its single strands are transferred and bonded to anitrocellulose membrane. strands are transferred and bonded to anitrocellulose membrane.

The Southern blot is used for genetic fingerprinting, diagnosis of infectious disease, The Southern blot is used for genetic fingerprinting, diagnosis of infectious disease, and other purposes. Also to demonstrate the incidence and prevalence in an and other purposes. Also to demonstrate the incidence and prevalence in an environmental sample of archaea, bacteria, and viruses, particularly those that environmental sample of archaea, bacteria, and viruses, particularly those that cannot be cultured. cannot be cultured.

Northen blot is a similar technique used to detect specific RNA molecule.Northen blot is a similar technique used to detect specific RNA molecule.

If the gel separates DNA and the DNA is detected with a DNA probe, it is called a If the gel separates DNA and the DNA is detected with a DNA probe, it is called a Southern blot. Southern blot.

If RNA is separated on the gel and then detected by a DNA probe, it is a Northen If RNA is separated on the gel and then detected by a DNA probe, it is a Northen blot. blot.

A Western blot uses specific antibodies to detect specific protein molecules on a A Western blot uses specific antibodies to detect specific protein molecules on a blot of a protein gel. In the Western blot, the role of the DNA probe is filled by an blot of a protein gel. In the Western blot, the role of the DNA probe is filled by an antibody that recognized a specific protein.antibody that recognized a specific protein.

Southern blot

technique

DNA MicroarraysDNA Microarrays

DNA microarrays consist of molecules DNA microarrays consist of molecules of single-stranded DNA that has been of single-stranded DNA that has been immoblized on glass slides, silicon immoblized on glass slides, silicon chips, or nylon membranes. chips, or nylon membranes.

Single strands of fluorescently-labeled Single strands of fluorescently-labeled DNA in a sample washed over an array DNA in a sample washed over an array adhere only to locations on the array adhere only to locations on the array where there are complementary DNA where there are complementary DNA sequences. sequences.

DNA microarrays can be used in a DNA microarrays can be used in a number of ways including monitoring number of ways including monitoring gene expression, diagnosing infection, gene expression, diagnosing infection, and identifying organisms in an and identifying organisms in an environmental sample.environmental sample.

UltracentrifugationUltracentrifugation Equilibrium density gradient Equilibrium density gradient

ultracentrifugation in CsCl is one of the ultracentrifugation in CsCl is one of the most commonly used DNA separation most commonly used DNA separation procedures. procedures.

The buoyant density of double stranded The buoyant density of double stranded Cs+ DNA depends on its base Cs+ DNA depends on its base composition, with DNAs of higher G + C composition, with DNAs of higher G + C content having a greater density. content having a greater density.

Single stranded DNA is ~0.015g cm-3 Single stranded DNA is ~0.015g cm-3 denser than the corresponding double denser than the corresponding double stranded DNA, so the two can be separated stranded DNA, so the two can be separated by equilibrium density gradient by equilibrium density gradient ultracentrifugation. ultracentrifugation.

Circular DNAs that are supercoiled to Circular DNAs that are supercoiled to different extents. different extents.