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microbial activity. Nitrosating agents can be formed

from certain compounds added to foods and as a

result of specific processing conditions. In most in-

stances, nitrosamines are found in foods in one of the

three following categories.

0016 Cured meats Nitrosamines are formed in cured

meats because nitrite, and sometimes nitrate, are

added to these products during processing. Nitrate is

reduced to nitrite by the enzyme nitrate reductase,

which occurs in a number of bacteria. As discussed

earlier, nitrite is converted to nitrosating agents which

subsequently react with amines in the meat during

processing, storage, and cooking to form nitros-

amines. (See Curing.)

0017 Nitrite and nitrate have been added to cured meats

for many years to prevent outgrowth and toxin for-

mation by Clostridium botulinum. Nitrite, in com-

bination with other curing ingredients such as sodium

chloride, is particularly effective in inhibiting forma-

tion of the deadly botulism toxin. In addition, nitrite

reacts with pigments in meat to impart the desirable

pink color of cured meats and it prevents the devel-

opment of off-flavors. (See Nitrates and Nitrites.)

0018 Although not all cured meats have detectable

amounts of nitrosamines, fried bacon has been

shown consistently to contain these compounds.

Typically, fried bacon contains 1–20 mgkg

1

NPYR and 1–3 mgkg

1

of NDMA. The formation is

related to the relatively high internal temperature of

bacon during frying and the relatively low moisture

content of bacon as compared to other cured meat

products. When bacon is cooked by other methods,

particularly in a microwave oven, considerably lower

amounts of nitrosamines are found. The majority of

evidence suggests that the free amino acid proline is

first nitrosated and then decarboxylated to form

NPYR during frying. Neither the precise chemical

nature of the nitrosating agent nor the amine precursor

for NDMA in bacon is known with certainty. How-

ever, the evidence suggests that the nitrosating agent is

a reaction product of nitrite and lipids in the bacon.

0019 Dried foods and ingredients A variety of processes

and equipment are used to dry foods and food ingre-

dients. During the direct-fire process, air used to dry

the food is first heated by passing the air through the

flames of burners. As a result, products of combus-

tion, including oxides of nitrogen, are directly incorp-

orated into the hot air used to dry the food. The

oxides of nitrogen which include nitrosating agents

such as nitrous anhydride can then react with amines

in the food being dried to produce nitrosamines.

0020 In 1979, scientists in Europe reported the occur-

rence of NDMA in beer. Soon after, reports from a

number of countries confirmed that most beers

contained 1–5 mgkg

1

of NDMA. Although beer

was found to contain NDMA, the NDMA was not

formed during the brewing process. Investigation led

to the discovery that direct-fire-dried malted barley,

an ingredient used in the manufacture of beer, was the

source of the NDMA.

0021A number of investigators have attempted to

identify the amine precursors of NDMA in malted

barley. The evidence suggests that dimethylamine

and perhaps the tertiary amine alkaloids gramine

and hordenine serve as precursors for NDMA in

malted barley.

0022The discovery of nitrosamine formation in direct-

fire-dried malted barley has led to the investigation of

other dried foods. In the USA, certain dried dairy

products, notably nonfat dry milk, are manufactured

by the direct-fire drying process. Nonfat dry milk

manufactured by this process consistently contains

low amounts (less than 1 mgkg

1

) of NDMA. Since

the direct-fire drying process is not used commonly

in Europe for manufacture of nonfat dry milk, the

product does not contain NDMA.

0023Seafood can contain nitrosamines as a result of

either cooking or salt-drying. When fish is broiled

with a gas flame, the oxides of nitrogen produced

in the flame can cause nitrosamine formation in a

manner analogous to nitrosamine formation in

direct-fire drying. In certain areas of the world, par-

ticularly the Orient, fish is preserved by salt. Often

sea salt is used which contains appreciable amounts

of nitrate. The nitrate is reduced to nitrite with subse-

quent formation of nitrosating agents. In some cases,

reaction with amines in the fish produces relatively

large amounts of nitrosamines, principally NDMA.

The potential for NDMA formation in fish is consid-

erable since high levels of NDMA precursors, such as

dimethylamine and trimethylamine, can occur in fish.

0024Migration from surfaces which contact foods Vul-

canized rubber products, such as baby nursing

nipples, have been shown to contain nitrosamines. It

has been demonstrated that when baby bottle nipples

are stored inverted in milk, the nitrosamines partially

migrate from the nipples to the milk. Furthermore,

studies have shown that nitrosamines migrate from

rubber netting used to hold cured meats during the

smoking process. In addition to rubber, nitrosamines

in such substances as wax-treated wrapping paper

and paperboard-based materials have been shown to

migrate to foods.

0025Nonvolatile nitrosamines in foods The notion

that nonvolatile nitrosamines might form in foods

follows logically from the fact that the precursors,

4144 NITROSAMINES

nonvolatile amine and nitrosating agents, occur in

foods. Due to limitations in analytical methodology

for nonvolatile nitrosamines, less is known about

the occurrence of nonvolatile than volatile nitrosa-

mines in foods. Recently developed methods for

nonvolatile nitrosamines have been limited to the

detection of N-nitrosated amino acids and amino

acid derivatives. Most analyses have been on

cured meats and include reports of compounds

such as NPRO, N-nitrososarcosine (NSAR),

N-nitroso-4-hydroxyproline, N-nitrosothiazolidine-

4-carboxylic acid, N-nitroso-2-methylthiazolidine-4-

carboxylic acid, and N-nitroso-2-hydroxyl-

methylthiazolidine-4-carboxylic acid. Other than

NSAR, which is a weak carcinogen, all the other N-

nitrosated amino acids and amino acid derivatives

which have been tested have not shown a carcino-

genic response in animals.

0026 Scientists conjecture that other nonvolatile nitros-

amines such as nitrosated peptides and nitrosated

amides occur in foods. Definitive information in

this regard awaits further application of analytical

methodology for nonvolatile nitrosamines to foods.

0027 Reduction of nitrosamine formation Considerable

efforts have been expended during the past 25 years

to reduce nitrosamine formation in foods. Scientists

have looked critically at the use of nitrite and nitrate

in the manufacture of cured meats. Since nitrite is

considered essential to guard against outgrowth and

toxin production by C. botulinum, its use in the

manufacture of cured meats has been retained. How-

ever, in many countries, permissible levels of nitrite

have been reduced to the minimum necessary for

control of botulism. Generally, nitrate is only permit-

ted in a few fermented cured meat products where

long-term inhibition of C. botulinum is required.

0028 The nitrosation inhibitors ascorbic acid and a-

tocopheral are either required or extensively used in

processing cured meats. Alternatives and/or partial

substitutes for nitrite in cured meats include use of

lactic acid-producing organisms, potassium sorbate,

sodium hypophosphite, fumarate esters, and ionizing

radiation. To date, no adequate alternative for nitrite

has been found. Consequently, nitrosamines continue

to be found in cured meats, especially fried bacon, but

generally at lower levels than occurred a number of

years ago.

0029 Efforts to reduce nitrosamine levels in barley malt,

and hence in beer, have been very successful. This

is largely due to conversion of direct-fired kilns to

indirect-fired kilns for the manufacture of barley

malt. With indirect-fired kilns, the products of com-

bustion are not incorporated into the drying air and,

therefore, nitrosamine formation in the barley malt is

greatly reduced. Interestingly, use of an indirect-fired

kiln does not completely inhibit nitrosation, probably

because ambient air which is drawn into the kiln

usually contains trace levels of oxides of nitrogen

and, therefore, some nitrosation occurs. In addition

to the use of indirect-fired kilns, sulfur dioxide, which

has been shown to be a nitrosation inhibitor, is some-

times used with both direct-fired kilns and indirect-

fired kilns to reduce nitrosamine formation.

0030Due to these changes in processing, the NDMA

levels in beer have been markedly reduced. In a recent

survey, Canadian and US beers were found to contain

on average 0.07 mgkg

1

of NDMA whereas, prior

to 1980, beer commonly contained 1–5 mgkg

1

NDMA.

0031Similarly, nitrosamine formation in rubber prod-

ucts, including baby nursing nipples, has been

reduced. This has been accomplished by substituting

nitrosatable with nonnitrosatable vulcanization

accelerator compounds in the manufacture of rubber

products.

0032Products other than food A variety of industrial,

agricultural, and consumer items have been shown to

contain nitrosamines. These include cosmetics, tobacco

products, industrial cutting fluids, vehicle tires, and

pesticides. In each case, the formation can be traced

to use of amines and contact with nitrosating agents

during product formulation, manufacture, or use.

0033Tobacco products are especially important in terms

of nitrosamine occurrence and subsequent human

exposure. During growth, tobacco plants biosynthe-

size a variety of alkaloids such as nicotine and nor-

nicotine. These compounds are either secondary or

tertiary amines. During curing, fermentation, and

aging the alkaloids react with nitrosating agents

formed from nitrate to produce a group of com-

pounds commonly referred to as tobacco-specific ni-

trosamines. Tobacco-specific nitrosamines have been

shown to occur in a wide range of tobacco products

such as cigarettes, cigars, pipe tobacco, chewing

tobacco, snuff, masheri, zarda, and nass. It is note-

worthy that the tobacco-specific nitrosamines fre-

quently occur in tobacco products at several orders

of magnitude higher than volatile nitrosamine occur-

rence in foods and beverages.

Endogenous Formation

0034Convincing evidence exists for endogenous formation

of nitrosamines in humans. Based on our knowledge

of nitrosation in acidic, aqueous media, it is not

surprising that nitrosamine formation has been dem-

onstrated to occur in the stomach. Estimation of

endogenous nitrosation in humans and experimental

animals has been accomplished by measuring urinary

NITROSAMINES 4145

NPRO following oral administration of proline and

nitrate. Inhibition of nitrosamine formation has been

accomplished by the administration of nitrosation

inhibitors such as ascorbic acid. Gastric nitrosation

proceeds through reaction between amines and nitro-

sating agents derived from the diet. Nitrate, which

occurs in substantial amounts in certain vegetables,

is partially reduced to nitrite by nitrate reductase-

containing bacteria in the oral cavity.

0035 Recently, the existence of endogenous nitrosation

pathways other than gastric nitrosation has been

recognized. Evidence exists for mammalian cellular

enzymatic conversion of arginine to nitric oxide,

which in turn affects nitrosation. Scientists believe

that this process occurs in several cell types, including

macrophages. The extent and relevance of cellular

nitrosation are yet to be determined.

Estimates of Exposure to Nitrosamines

from Foods

0036 Several groups have made estimates of human expos-

ure to volatile nitrosamines from food and beverage

consumption. Most of the estimates relate to expos-

ure from consumption of foods and beverages in

western Europe. Volatile nitrosamine exposure was

predominately from NDMA and NPYR.

0037 The estimation of daily exposure to volatile nitros-

amines in most reports ranged from 0.1 to 1.0 mg per

person. In comparison, a US National Academy of

Sciences report in 1981 estimated exposure to nitros-

amines from cigarette smoking to be 17 mg per person

per day.

0038 Several recent reviews reported an estimated daily

exposure to nitrosamines of 10–120 mg per person

from diet. Estimates in this range include, in addition

to volatile nitrosamines, N-nitrosated amino acids

and amino acid derivatives and some include appar-

ent total N-nitroso compounds as well. It should be

recognized that most N-nitrosated amino acids and

amino acid derivates have failed to elicit a carcino-

genic response in animals and the identity and

carcinogenicity of the apparent total N-nitroso com-

pounds are unknown.

0039 Some cautionary comments are needed regarding

estimates of exposure to nitrosamines. First, estimates

should be based on nitrosamines with known identity

and known carcinogenicity in animals. Second, a

number of the estimates were conducted a decade

ago or are based on reports of the volatile nitrosamine

content of foods from over a decade ago. As discussed

previously, the volatile nitrosamine content of foods,

e.g., NDMA in beer, has been dramatically reduced in

recent years. Therefore, some of the estimates in the

literature may be higher than reflected by current

levels of volatile nitrosamines. Third, information

currently available does not allow reliable estimation

of exposure from currently undetected carcinogenic

nonvolatile nitrosamines, of nitrosamines formed

endogenously, and of other N-nitroso compounds

formed exogenously and endogenously. A fourth limi-

tation of estimates is that they are based on average

food consumption for relatively large populations.

Food consumption patterns for subgroups and indi-

viduals within populations vary widely and, there-

fore, so does exposure to nitrosamines in the diet.

Role of Nitrosamines and Other

-Nitroso

Compounds in Human Cancer

0040Based on current information on carcinogenicity in

experimental animals, and on other pertinent infor-

mation, most scientists believe that nitrosamines and

other N-nitroso compounds are capable of inducing

cancer in humans. However, at the present time we do

not know what exposure to the various nitrosamines

is required to induce cancer in a human population.

0041Convincing evidence exists for the role of tobacco-

specific nitrosamines in cancer induction in people

who use tobacco products. Particularly compelling

evidence exists for the causative role of tobacco-

specific nitrosamines in cancer of the oral cavity

for people who engage in snuff dipping and chewing

betel quid.

0042In order to assess more fully the part nitrosamines

and other N-nitroso compounds play in human

cancer, progress will be needed in the following

areas: improvement and application of analytical

methodology will be required for estimation of

exposure to nonvolatile nitrosamines and other

N-nitroso compounds in foods and in other materials;

second, a better understanding of formation and

exposure to endogenously formed nitrosamines and

other N-nitroso compounds is needed; and, finally,

methodology will need to be improved in order to

allow reliable estimation of human cancer incidence

from exposure to relatively low levels of carcinogens,

including nitrosamines.

See also: Amines; Cancer: Carcinogens in the Food

Chain; Carcinogens: Carcinogenic Substances in Food:

Mechanisms; Carcinogenicity Tests; Clostridium:

Occurrence of Clostridium botulinum; Botulism; Curing;

Drying: Equipment Used in Drying Foods; Nitrates and

Nitrites; Smoking, Diet, and Health

Further Reading

Forman D and Shuker D (eds) (1989) Cancer Surveys,

Advances and Prospects in Clinical, Epidemiological

and Laboratory Oncology. Nitrate, Nitrite and

4146 NITROSAMINES

Nitrosocompounds in Human Cancer, vol. 8, no. 2.

Oxford: Oxford University Press (published for the

Imperial Cancer Research Fund).

Gloria MBA, Barbour JF and Scanlan RA (1997) N-Nitro-

sodimethylamine in Brazilian, US domestic, and US

imported beers. Journal of Agriculture and Food

Chemistry 45: 814–816.

Gloria MBA, Barbour JF and Scanlan RA (1997) Volatile

nitrosamines in fried bacon. Journal of Agriculture and

Food Chemistry 45: 1816–1818.

Hill MJ and Giacosa A (eds) (1996) Proceedings of the

thirteenth annual ECP symposium N-Nitroso com-

pounds in human cancer: current status and future

trends. European Journal of Cancer Prevention 5 (sup-

plement 1): 1–163.

Hotchkiss JH (1989) Relative exposure to nitrite, nitrate

and N-nitroso compounds from endogenous and

exogenous sources. In: Taylor SL and Scanlan RA (eds)

Food Toxicology, a Perspective on the Relative Risks.

IFT Basic Symposium Series. New York: Marcel Dekker.

O’Neill IK, Chen J and Bartsch H (eds) (1991) Relevance to

Human Cancer of N-Nitroso Compounds, Tobacco

Smoke and Mycotoxins. IARC Scientific Publication,

no. 105. Lyon: International Agency for Research on

Cancer.

Scanlan RA and Tannenbaum SR (eds) (1981) N-Nitroso

Compounds. ACS Symposium Series, no. 174. Washing-

ton, DC: American Chemical Society.

Tricker AR (1997) N-nitroso compounds and man: sources

of exposure, endogenous formation and occurrence in

body fluids. European Journal of Cancer Prevention 6:

226–268.

Tricker AR and Preussmann R (1991) Carcinogenic N-

nitrosamines in the diet: occurrence, formation, mech-

anisms and carcinogenic potential. Mutation Research

259: 277–289.

NMR Spectroscopy See Spectroscopy: Overview; Infrared and Raman; Near-infrared; Fluorescence;

Atomic Emission and Absorption; Nuclear Magnetic Resonance; Visible Spectroscopy and Colorimetry

Nonstarch Polysaccharides See Dietary Fiber: Properties and Sources; Determination;

Physiological Effects; Effects of Fiber on Absorption; Bran; Energy Value

NUCLEIC ACIDS

Contents

Properties and Determination

Physiology

Properties and Determination

D W Gruenwedel, University of California at Davis,

Davis, CA, USA

This article is reproduced from Encyclopaedia of Food Science,

Presence of Nucleic Acids in Food

0001 Nucleic acids are natural constituents of all foods

derived from animal or plant sources, for they are

intrinsic components of the cells making up food

tissue. They occur usually in close association with

basic proteins (histones, protamines), forming nucleo-

proteins. In general, their effect on texture, nutritive

value, or sensory properties of foods is small in view

of their low tissue concentrations (typically < 200 mg

of nucleic acid phosphorus per 100 g of fresh tissue).

These low tissue concentrations normally also pre-

clude the occurrence of detrimental health effects.

However, health consequences may arise with per-

sons suffering from metabolic disorders such as im-

pairment of purine excretion. In these instances, the

dietary breakdown of the nucleic acids can generate

NUCLEIC ACIDS/Properties and Determination 4147

unacceptably high levels of uric acid in blood as well

as tissues, producing ultimately hyperuricemia, acute

and chronic arthritis, or other symptoms of gout.

Properties of Nucleic Acids

Chemical Properties

0002 Nucleic acids are macromolecules of high molecular

weight, made up of heterocyclic pyrimidine (Py) and

purine (Pu) bases, a five-membered (furanose-type)

sugar ring, and orthophosphate. Bases, sugar and

phosphate occur in equimolar ratios, i.e., Pu(Py):

sugar: phosphate of 1:1:1. Thus, schematically, their

chemical composition may be denoted as

½PuðPyÞpentose  PO



n, ð1Þ

with n being a large number. Among naturally

occurring nucleic acids, n may range from 4.5 10

(polyoma virus) to 7.0 10

(Drosophila melanoga-

ster chromosome). Two broad categories exist, de-

oxyribonucleic acids (DNAs) and ribonucleic acids

(RNAs), depending on whether the sugar moiety

is b-d-2

-deoxyribose or b-d-ribose (note: primed

entities refer to sugar-binding sites). Their elemental

composition is roughly 15% nitrogen, 10% phos-

phorus, 36% carbon, 4% hydrogen, and 35%

oxygen. Cellular DNA is localized almost exclusively

in the nucleus, while RNA is found predominantly in

the cytoplasm.

0003 The principal bases found associated with DNAs

are the purine derivatives adenine (6-aminopurine,

Ade) and guanine (2-amino-6-hydroxypurine, Gua),

and the pyrimidine derivatives cytosine (2-hydroxy-4-

amino-pyrimidine, Cyt) and thymine (5-methyluracil,

Thy). Occasional base variations (e.g., 5-methylcyto-

sine or 5-hydroxymethylcytosine) are encountered.

In RNAs, the base Thy is replaced by uracil (2,4-

dihydroxypyrimidine, Ura). Certain RNAs, e.g.,

(transfer) tRNAs, show considerable base variations:

they may contain a wide variety of methylated bases,

including Thy, bases with thio groups, inosine, etc.

0004 The Pu bases are bound to the sugar in an

N9 !C1

-glycosidic linkage; with Py bases, bonding

occurs via Nl !Cl

. The resulting compounds are

N-glycosides; they are called nucleosides. Some

nucleosides are: adenosine (Ado or A), guanosine

(Guo or G), cytidine (Cyd or C), uridine (Urd or U),

and thymidine (dThd or dT). Attachment of ortho-

phosphate in positions O3

(or O5

) produces the

corresponding 3

(or 5

) (mono)nucleotides. 5

-Mono-

nucleotides are the actual building blocks of DNAs

and RNAs. Schematically, their polymerization

takes place by 5

phosphodiester bond forma-

tion, e.g., the 5

-phosphate group of one nucleotide

links up with the free 3

-hydroxyl group of another,

thereby forming a dinucleotide. It is customary to

use short-hand notations such as 5

-pApCpGpT-3

or 5

-ApCpGpTp-3

to indicate 5

-terminal and 3

terminal phosphate, respectively.

0005In both DNA and RNA, in-vivo diester bond for-

mation proceeds solely in the 5

! 3

direction, pro-

ducing long-chain, unbranched structures (strands).

Branching in RNA molecules can occur transiently

during RNA splicing. Both DNA and RNA strands

display polarity, meaning that their primary structure

is to be read in the direction ! O3

! [PO

! O5

! C4

! C3

! O3

] ! [PO

! O5

! C5

! C3

! O3

], etc. (the brackets define a DNA/

RNA monomer subunit). The strand polarity is a

consequence of furanose pucker as well as associated

base stacking.

0006The chemical, i.e., base composition of nucleic

acids is variable and differs from one species to an-

other. Variation in base composition forms the basis

of the genetic information content of DNAs. How-

ever, in DNAs, certain regularities apply (Chargaff’s

rules): (1) ~Pu ¼~Py, (2) ~A ¼~T and ~G ¼~C,

(3) ~(A þ C) ¼~(G þ T). On occasion, 5-methyl-

cytosine needs to be taken into consideration, too, in

order to satisfy points (2) and (3). These rules hold

because of the double-stranded (complementary)

structure of DNAs brought about, in part, by

Watson–Crick hydrogen bonding between opposite

bases (A

—

T and G

—

C base pair formation; the

symbols

—

and

—

denote that base pairing between

A and T involves two hydrogen bonds, while three

hydrogen bonds are formed in the base pairing

between G and C). Chargaff’s rules do not apply to

single-stranded nucleic acids, whether they are RNAs

(usually) or DNAs (occasionally).

Physiochemical Properties

0007At physiological pH values, the orthophosphate

groups of the nucleic acids are negatively charged.

Outward charge neutralization occurs through posi-

tively charged counterions aligning themselves with

the negative charges on the polymers. Counterions

can be Na

,Mg

2þ

, basic proteins, polyamines, etc.

Nucleic acids are therefore polyelectrolytes, a prop-

erty that determines their solution behavior: they are

extensively hydrated and form gel-like structures if

present in higher concentrations. Even in dilute solu-

tions they display non-Newtonian behavior, as noted,

for instance, by their shear-rate-dependent viscosity.

Once hydrated, they are readily soluble in water.

Isolation of nucleic acids from tissues usually yields

their sodium salt. How they really exist in vivo is

unknown, although it is assumed that Na

also serves

there as the principal counterion.

4148 NUCLEIC ACIDS/Properties and Determination

0008 DNAs, with very few exceptions, are double-

stranded helices of opposite polarity. By contrast,

RNAs are usually single-stranded, although double-

(as well as triple-) stranded RNAs are known to exist.

There are also duplex helices made up of DNA and

RNA single strands; they form, for example, in vivo

during transcription. DNA double-strandedness,

brought about by interstrand hydrogen bonding and

intrastrand base stacking, is termed DNA secondary

structure; this structure is salient to the preservation

of genetic information by DNA. Hydrophobic bond-

ing and London dispersion forces are the major

factors contributing to base stacking. DNA secondary

structure is quite stable over a wide range of pH

(3–12), ionic strength, I (0.001–5), and temperature

(up to 100



C). The ranges given are approximate: not

only are the effects of I and temperature interdepend-

ent (rule-of-thumb: the higher I is, the higher the

thermal stability), but base composition (GC-rich

DNAs are more heat-stable than AT-rich DNAs) con-

tributes heavily to the stability of DNA secondary

structure as well. Lastly, at elevated levels of I, indi-

vidual cation/anion effects, often destabilizing in

nature, also become noticeable. Transforming the

DNA double helix into two single strands of DNA,

a process occurring cooperatively, is called ‘denatur-

ation.’ Synonymous terms are ‘helix-to-random coil

transition’ or ‘order–disorder transition.’‘Denatur-

ation’ represents a change in DNA conformation

(conformational changes are those that rupture

hydrogen bonds and/or hydrophobic bonds but

leave covalent bonds intact). It is evident from the

above that variations in pH, I, and temperature can

be used to bring about changes in DNA conformation

experimentally. While much is known regarding the

subtleties of DNA secondary structure, very little in-

formation is available with respect to the structural

properties of the single-stranded states of DNA (or

RNA).

0009 Nucleic acids are optically active molecules, i.e.,

they rotate the plane of (linearly) polarized light

and, hence, display chirality. The major contribution

to optical activity comes from base stacking, for it

imparts molecular asymmetry. The optical activity of

the asymmetric carbon atoms of deoxyribose or

ribose does not contribute much to the overall effect.

Double-stranded DNAs are usually of right-handed

screwness or helicity. This information has been

obtained from X-ray diffraction analysis of DNA

fibers. Depending on the sign and magnitude of the

parameters defining the orientation of a base pair in

DNA relative to the helix axis (e.g., rotational twist,

t, tilt, y

, roll y

, propeller twist, y

, axial rise, h,

pitch, width, and depth of major and minor grooves),

one can distinguish between conformational families

such as A-, B-, C-, and Z-type DNA. They exist

among naturally occurring DNAs, i.e., DNAs with

random base sequences. Synthetic DNAs (poly-

nucleotides with nonrandom sequences) are charac-

terized by additional families such as B

,D,E,

S, Z

. Z-type DNA is a left-handed double helix.

0010Until quite recently, the consensus was that

double-stranded DNA is rather inflexible (‘rod’ or

‘worm-like chain’) and that, apart from its exciting

base-pairing capabilities, the structure of the sugar–

phosphate backbone, in view of its seeming monot-

ony, is of little consequence to its biological function.

This perception was due to the fact that X-ray diffrac-

tion studies executed on DNA fibers gave structural

information only at low levels of resolution. How-

ever, experimental evidence has shown that double-

stranded DNA is surprisingly flexible locally, giving

rise to what has been termed DNA ‘polymorphism.’

DNA polymorphism refers to seemingly minor, quite

localized alterations in secondary structure that may

very well be of crucial importance in biological pro-

cesses such as replication and transcription. That

there is such a thing as DNA polymorphism has

been demonstrated by X-ray diffraction analysis of

oligonucleotide crystals. Oligonucleotide crystals

(formerly not available) yield high-resolution X-ray

data down to atomic dimensions. It was found that

DNA polymorphism resides in the nonplanar ring

structure of b-d-2

-deoxyribose (or b-d-ribose in

RNA) and that this structure is conformationally

quite flexible. Its two main conformations (called

sugar pucker) are envelope E (four atoms in a plane,

the fifth out of plane) and twist T (three atoms in a

plane, the two others out of plane on opposite sides).

Out-of-plane atoms on the same side of C5

are de-

noted endo, while those on the opposite side are

denoted exo. Transitions between E and T are facile,

giving rise to a pseudorotation cycle of the furanose

ring in nucleosides. The cycle contains 10 different

(endo/exo) T and E forms. In addition, O5

can

assume a number of orientations about the C4

–C5

bond axis; they are known as gauche, gauche

(þsynclinal); gauche, trans (antiperiplanar); and

trans, gauche (synclinal). Lastly, relative to the

sugar moiety, a Pu can adopt two major orientations

along the N—Cl

bond; anti (the bulk of the base

turns away from the sugar) and syn (it is over or

toward the sugar). All orientations influence each

other, giving rise to a multitude of sugar conform-

ations – and, hence, structural DNA families – with

important implications for biological function. Thus,

the binding of enzymes (e.g., DNase I, restriction

endonucleases) or regulatory proteins such as TFIIIA

(zinc fingers) to DNA is now known to be greatly

influenced by the geometry of the backbone. In fact,

NUCLEIC ACIDS/Properties and Determination 4149

it has been said that while base pairing may be viewed

as the ‘brain’ of DNA, the conformational flexibility

of the sugar–phosphate backbone constitutes its

‘heart.’

Determination and Characterization of

Nucleic Acids

0011 There exist literally hundreds of techniques that

permit the detection, quantitative evaluation, struc-

tural, and biological characterization of nucleic acids.

They are chemical, biochemical, physicochemical, or

physical in nature; however, their description is

beyond the scope of this article. ‘Determination of

nucleic acids’ is therefore understood here to mean

their detection (qualitatively as well as quantitatively)

in cells and tissues in particular, and the determin-

ation of their chemical composition (average base

composition) when in solution. Hence, techniques

that serve their physical characterization in terms of

structure, mass, or shape, or techniques used in DNA

sequencing, in the enzymatic manipulation of DNA

and RNA, in recombinant DNA research, in cloning,

etc., are not presented. Techniques relating to their

isolation from animal and plant tissues or from

microbial sources are also not discussed here.

Detection of Nucleic Acids in Cells and Tissues

0012 Histochemical approaches (in-situ staining) Most

advantageous are histochemical staining techniques.

Each constituent of DNA/RNA (cf. eqn (1)) can be

identified histochemically: acidic phosphate groups

are demonstrated with the aid of basic dyes, b-d-2

deoxyribose with the help of a triphenylmethane dye,

and Pu(Py) can be made visible via intercalation with

fluorescent dyes. The specificity of the techniques can

be improved further through judicious use of DNA

(deoxyribonuclease) and RNA (ribonuclease) degrad-

ing enzymes prior to staining. This eliminates possible

interference by the other polymer. Although staining

techniques are usually used to obtain qualitative

information, they can be adapted to furnish also

quantitative data.

0013 Techniques detecting DNA

Feulgen nucleal reaction DNA in tissue is depuri-

nated by strong acid treatment. The resulting apurinic

acid releases b-d-2

deoxyribose, which in turn is

converted to its open-chain aldehyde form. The

sugar aldehyde reacts with the colorless triphenyl-

methane derivative leucofuchsin (Schiff’s reagent) to

give a magenta color (fuchsin). The chemistry of the

reaction is known. Since the ribose of RNA cannot be

changed to an open-chain aldehyde form, RNA will

not be stained; hence, this technique is very specific.

0014Kurnick’s methyl green method Methyl green, a tri-

phenylmethane dye possessing two positively charged

amine groups, adds on to double-stranded DNA.

Details of the process remain obscure but appear to

be related to the secondary structure of DNA: the two

positively charged amine groups of the dye are said to

have just the correct distance to add on to phosphate

groups separated from one another by one turn of the

DNA double helix. DNA stains green or green-blue.

0015Fluorescence flow cytometry A number of macro-

cyclic dyes (acridine orange (derivative of acridines),

Hoechst 33342, Hoechst 33258 (derivatives of

piperazine), ethidium, propidium (derivatives of phe-

nanthridine), etc.) bind to DNA bases. Some of them

(e.g., acridine orange) will also react with RNA. Their

commonality resides in the fact that they are planar,

highly aromatic molecules that fluoresce when irradi-

ated. Because of their flatness, they can bind to DNA

through intercalation, i.e., they can sandwich them-

selves between bases. Intercalation increases their

basic fluorescence, which makes the nucleic acids

visible. Excitation sources are mercury arc lamp,

argon ion laser, or krypton ion laser. Since DNA–

dye emission spectra differ usually from those of the

RNA–dye complexes, distinction between the two

types of nucleic acids is possible. Determination of

DNA (or RNA) is quantitative. The technique is used

most frequently in cell-culture work or in studies in

which free-flowing cellular or subcellular particles

are available (e.g., red blood cells).

0016Techniques detecting DNA and RNA

Methyl green–pyronin method If proper experimen-

tal conditions are maintained, nuclei (DNA) will be

stained green or blue (methyl green), and nucleoli

and cytoplasm (RNA) red (pyronin). Since pyronin,

a xanthene derivative, is also a fluorescent dye, the

technique can be adopted for flow-cytometric

determination of RNA.

0017Acridine orange method The dye acridine orange

binds to DNA and RNA. The two dye–polymer com-

plexes exhibit different fluorescence emission spectra:

when illuminated with purple light, DNA fluoresces

green–yellow to bright yellow and RNA reddish

brown to orange; when illuminated with ultraviolet

light, DNA fluoresces greenish yellow and RNA crim-

son red. (See Spectroscopy: Fluorescence.)

0018Nonhistochemical Approaches

Radioactive labeling method Nucleic acids can be

further identified qualitatively or quantitatively in

cells or tissues by labeling them radioactively. Fre-

quently used radionuclides are

C, and

4150 NUCLEIC ACIDS/Properties and Determination

They are administered experimentally in the form of

labeled precursors of DNA or RNA (e.g., [

H]dT

for DNA, [

H]U for RNA, [ a-

P]dATP for DNA,

[a-

P]ATP for RNA, etc.) and are incorporated

into the polymers enzymatically during macromol-

ecular synthesis. Labeling can be performed in vitro

or in vivo. The presence of the radionuclides and,

hence, of the nucleic acids is noted by the radioactive

decay of the nuclides in the form of a or b particles

or g rays.

0019 Burton assay for DNA This assay is a colorimetric

procedure for measuring the deoxyribose moiety of

diphenylamine DNA. Color is produced by a second-

ary amine (phenylamine) reacting with the sugar.

The assay is relatively specific, although RNA in

high concentrations, or sucrose, should be absent.

DNA does not have to be present in purified form;

thus, determinations can be undertaken in crude cel-

lular extracts. DNA is usually extracted from tissue or

cells through treatment with strong acids (perchloric

acid, trichloroacetic acid), although alkaline extrac-

tions have also been used. Extraction is undertaken to

remove interfering substances, particularly proteins.

Color readings are taken at 595 and 650 nm. A stand-

ard curve is prepared by using samples of pure DNA

(commercially available). A number of modifications

of the basic procedure exist. (See Spectroscopy:

Visible Spectroscopy and Colorimetry.)

0020 3,5-Diaminobenzoic acid fluorescence assay DNA

is extracted from cellular material with acid treat-

ment (usually perchloric acid) and the sample ex-

posed to hydrochloric acid (1 N) for about 45 min

at 55–57



C. This produces apurinic acid and free

b-d-2

-deoxyribose (see section Feulgen nucleal reac-

tion). The C1

and C2

carbons of the sugar then react

with 3,5-diaminobenzoic acid to produce a strongly

fluorescent compound. Excitation occurs at 410 nm;

fluorescence is measured at 510 nm. Calibration with

pure DNA is required. Submicrogram quantities of

DNA can be determined.

Detection of Nucleic Acids in Solution

0021 These techniques assume DNA as well as RNA to

exist in solution in essentially pure form, i.e., extrac-

tion from tissues or cells and purification occurring

prior to analysis.

0022 Spectrophotometric methods

Spectrophotometry in the ultraviolet (concentration

determinations) The presence of stacked Pu(Py)

bases in DNA/RNA makes the polymers absorb radi-

ation in the ultraviolet region. With duplex DNA,

absorption starts at wavelengths below 300 nm and

reaches a maximum near 260 nm and a minimum

around 230 nm. The presence of a second peak around

200 nm has been suggested; however, its demonstra-

tion is not simple because most spectrophotometers

cease functioning around 200 nm. Concentration

determinations are usually performed at 260 nm by

applying the Bouguer–Lambert–Beer law:

260

¼ e

260

cd, ð2Þ

where A

260

is the absorbance of DNA at 260 nm, e

260

the DNA molar absorptivity (1 (mol phosphate)

1

), c its molar concentration in solution (mol

phosphate l

1

), and d the optical pathlength (cm).

By measuring A

260

, one can readily compute c, the

desired quantity, e

260

, and these are available from the

literature for almost all DNAs. They are usually

around 6.6 10

l (mol phosphate)

1

. Because

of the 1:1:1 molar relationship between phosphate,

sugar, and base, c can also be expressed in terms of

moles of base per liter or moles of monomer per liter,

etc. Since the average molecular weight of a nucleic

acid monomer unit is 330, it is easily derived from

eqn (2) that for duplex DNA:

1 A

260

unit ¼ 50 mg DNA ml

1

, ð3Þ

while for single-stranded DNA or for RNA

1 A

260

unit ¼ 40 mg RNA ml

1

: ð4Þ

0023Spectrophotometric methods in the ultraviolet (base

composition determinations) The secondary struc-

ture of DNA can be used for analytical purposes.

Heating duplex DNA at a given pH and ionic strength

ultimately yields single-stranded DNA (‘denatur-

ation’). The process can be followed spectrophotome-

trically since single-stranded DNA has a higher

absorbance (by about 25% at room temperature) at,

say, 260 nm than double-stranded DNA. The increase

in absorbance is called ‘hyperchromicity’ (H) and is

defined as follows:

260

¼ A

260

 A

260



260

, ð5Þ

where A

260

is the absorbance of DNA at 260 nm at a

given temperature, t, while A

is its absorbance prior

to heating (usually room temperature). H is thus the

normalized absorbance increase of DNA due to heat-

induced denaturation. Plotting H

260

against tempera-

ture produces the so-called DNA ‘melting curve.’ The

expression ‘melting’ refers to the fact that DNA

‘helix-to-random coil transitions’ resemble the infin-

itely sharp temperature-dependent phase transitions

occurring in ice during melting. DNA ‘melting curves’

are of sigmoidal shape. The limiting value of A

260

NUCLEIC ACIDS/Properties and Determination 4151

max

is about 40% higher than A

260

. The temperature

at which H

260

amounts to 50% of its limiting value

260

max

is called the ‘melting temperature’ (T



C).

is linearly related to the (average) base compos-

ition of DNA. For the ‘standard saline citrate’

medium (0.15 M sodium chloride and 0.0015 M

sodium citrate, pH 7.0):

%GC ¼ 2:44 ðT

 69 :3 Þ:ð6Þ

Since % GC ¼ 100X

, with X

, representing the

‘mole fraction’ of GC base pairs in DNA, and since

¼ 1  X

, eqn (6) furnishes equally well infor-

mation on %AT. Equations similar to eqn (6) have

been established for other solvents. In conclusion,

heating native DNA under controlled conditions

(constant pH and ionic strength, application of a

linear temperature gradient, automatic recording of

hyperchromicity) enables the rapid determination

of its chemical (base) composition. It is best to work

at A

260

 1( 50 mg DNA ml

1

Seealso: Spectroscopy:Fluorescence;Visible

SpectroscopyandColorimetry