Nucleic acid

NUCLEIC ACID
VIJAY
JRF
GITAM UNIVERSITYDr. Wolf's CHM 424 28- 1

Pyrimidines and Purines
• In order to understand the structure and properties
of DNA and RNA, we need to look at their structural
components.
• We begin with certain heterocyclic aromatic
compounds called pyrimidines and purines.
Dr. Wolf's CHM 424 28- 2

• Pyrimidine and purine are the names of the
parent compounds of two types of nitrogen-
containing heterocyclic aromatic compounds.
PyrimidinePyrimidine PurinePurine
NN
NN NN
HH
NN
NNNN 11
22
33
44
5577
88
99
11
22
33
44
55
66
66

• Amino-substituted derivatives of pyrimidine and
purine have the structures expected from their
names.
4-Aminopyrimidine4-Aminopyrimidine 6-Aminopurine6-Aminopurine
HH
NN NN
NNNN
HH
HH
NNHH22
NN
NN
HH
HH
HHHH22NN

• But hydroxy-substituted pyrimidines and purines
exist in keto, rather than enol, forms.
enolenol
NN
NN
HH
HH
HHHHOO
NN
NN
HH
HH
HHOO
HH
ketoketo

• But hydroxy-substituted pyrimidines and purines
exist in keto, rather than enol, forms.
HH
NN NN
NNNN
HH
HH
OOHH
enolenol ketoketo
HH
NN NN
NN
HH
HH
OO
HHNN

Important Pyrimidines
• Pyrimidines that occur in DNA are cytosine and
thymine. Cytosine and uracil are the pyrimidines
in RNA.
HHNN
NN
HH
OO
OO
UracilUracil
HHNN
NN
HH
OO
OO
CHCH33
ThymineThymine
HHNN
NN
HH
NNHH22
OO
CytosineCytosine

Important Purines
• Adenine and guanine are the principal purines of
both DNA and RNA.
AdenineAdenine
NN
NN
NNHH22
NN
NN
HH
GuanineGuanine
OO
HHNN
NN
HH
NN
NN
HH22NN

Caffeine and Theobromine
• Caffeine (coffee) and theobromine (coffee and
tea) are naturally occurring purines.
CaffeineCaffeine
NN
NN
OO
NN
NN
HH33CC
OO
CHCH33
CHCH33
TheobromineTheobromine
OO
HHNN
NNNN
NN
CHCH33
CHCH33
OO

Nucleosides
• The classical structural definition is that a nucleoside
is a pyrimidine or purine N-glycoside of D-
ribofuranose or 2-deoxy-D-ribofuranose.
• Informal use has extended this definition to apply to
purine or pyrimidine N-glycosides of almost any
carbohydrate.
• The purine or pyrimidine part of a nucleoside is
referred to as a purine or pyrimidine base.

Table 28.2
• Pyrimidine nucleosides
NNHH22
HOHO OHOH
OOHOCHHOCH22
OO
NN
NN
Cytidine occurs in RNA;Cytidine occurs in RNA;
its 2-deoxy analog occurs in DNAits 2-deoxy analog occurs in DNA
CytidineCytidine

Table 28.2
OO
NN
NNHH
OO
HOHO
OO
HH33CC
HOCHHOCH22
Thymidine occurs in DNAThymidine occurs in DNA
ThymidineThymidine

Table 28.2
HOCHHOCH22
OO
NN
NNHH
OO
OHOHHOHO
OO
Uridine occurs in RNAUridine occurs in RNA
UridineUridine

Table 28.2
• Purine nucleosides
AdenosineAdenosine
Adenosine occurs in RNA;Adenosine occurs in RNA;
HOCHHOCH22 OO
OHOHHOHO
NN
NN
NN
NHNH22
NN

Table 28.2
• Purine nucleosides
GuanosineGuanosine
Guanosine occurs in RNA;Guanosine occurs in RNA;
HOCHHOCH22
NN
NHNH
OO
OO
HOHO
NN NHNH22
NN
OHOH

Adenosine 5'-Monophosphate (AMP)
• Adenosine 5'-monophosphate (AMP) is also called
5'-adenylic acid.
OCHOCH22PPHOHO
OO
HOHO
OO
OHOHHOHO
NN
NN
NN
NHNH22
NN
5'5'
1'1'
2'2'3'3'
4'4'

Adenosine Diphosphate (ADP)
OOPP
OO
HOHO
HOHO OCHOCH22PP
OO
HOHO
OO
OHOHHOHO
NN
NN
NN
NHNH22
NN

Adenosine Triphosphate (ATP)
• ATP is an important molecule in several
biochemical processes including:
energy storage (Sections 28.4-28.5)
phosphorylation
OOPP
OO
HOHO
OO OCHOCH22PP
OO
HOHO
OO
OHOHHOHO
NN
NN
NN
NHNH22
NN
PP
OO
HOHO
HOHO

ATP and Phosphorylation
ATPATP ++
hexokinasehexokinaseThis is the first step in theThis is the first step in the
metabolism of glucose.metabolism of glucose.
ADPADP ++
OO
OHOH
HOHO
HOHO
HOHO
(HO)(HO)22POCHPOCH22
OO
HOCHHOCH22
OO
OHOH
HOHO
HOHO
HOHO

cAMP and cGMP
• Cyclic AMP and cyclic GMP are
"second messengers" in many
biological processes. Hormones
(the "first messengers")
stimulate the formation of
cAMP and cGMP.
Cyclic adenosine monophosphate (cAMP)Cyclic adenosine monophosphate (cAMP)
CHCH22 OO
OHOH
NN
NN
NN
NHNH22
NN
OO
OO
PP
OO
HOHO

cAMP and cGMP
• Cyclic AMP and cyclic GMP are
"second messengers" in many
biological processes. Hormones
(the "first messengers")
stimulate the formation of
cAMP and cGMP.
OO
OO
PP
OO
HOHO
Cyclic guanosine monophosphate (cGMP)Cyclic guanosine monophosphate (cGMP)
CHCH22
NN
NHNH
OO
OO
NN NHNH22
NN
OHOH

Bioenergetics
• Bioenergetics is the thermodynamics of biological
processes.
• Emphasis is on free energy changes (∆G)
• when ∆G is negative, reaction is
spontaneous in the direction written
• when ∆G is 0, reaction is at equilibrium
• when ∆G is positive, reaction is not
spontaneous in direction written

Standard Free Energy (∆G°)
• Sign and magnitude of ∆G depends on what the
reactants and products are and their
concentrations.
• In order to focus on reactants and products,
define a standard state.
• The standard concentration is 1 M (for a reaction
in homogeneous solution).
• ∆G in the standard state is called the standard
free-energy change and given the symbol ∆G°.
mmA(A(aqaq)) nnB(B(aqaq))

• Exergonic: An exergonic reaction is one for which
the sign of ∆G° is negative.
• Endergonic: An exergonic reaction is one for
which the sign of ∆G° is positive.

• It is useful to define a special standard state for
biological reactions.
• This special standard state is one for which the
pH = 7.
• The free-energy change for a process under
these conditions is symbolized as ∆G°'.

Hydrolysis of ATP
• ∆G°' for hydrolysis of ATP to ADP is –31 kJ/mol
• Relative to ADP + HPO4
2–
, ATP is a "high-energy"
compound.
• When coupled to some other process, the
conversion of ATP to ADP can provide the free
energy to transform an endergonic process to an
exergonic one.
ATP + HATP + H22OO ADP + HPOADP + HPO44
2–2–

Glutamic Acid to Glutamine
++ NNHH44
++––
OCCHOCCH22CHCH22CHCOCHCO––
++
NHNH33
OO OO
+ H+ H22OOHH22NNCCHCCH22CHCH22CHCOCHCO––
++
NHNH33
OO OO
∆∆GG°' = +14 kJ°' = +14 kJ Reaction is endergonicReaction is endergonic

++ NNHH44
++––
++
NHNH33
OO OO
Reaction becomes exergonicReaction becomes exergonic
when coupled to the hydrolysiswhen coupled to the hydrolysis
of ATPof ATP
+ ATP+ ATP
+ HPO+ HPO44
2–2–
HH22NNCCHCCH22CHCH22CHCOCHCO––
++
NHNH33
OO OO
∆∆GG°' = –17 kJ°' = –17 kJ
+ ADP+ ADP

––
++
NHNH33
OO OO
Mechanism involvesMechanism involves
phosphorylation of glutamicphosphorylation of glutamic
acidacid
+ ATP+ ATP
OOCCHCCH22CHCH22CHCOCHCO––
++
NHNH33
OO OO
+ ADP+ ADPPP
OO
––
OO
––
OO

followed by reaction offollowed by reaction of
phosphorylated glutamic acidphosphorylated glutamic acid
with ammoniawith ammonia
HH22NNCCHCCH22CHCH22CHCOCHCO––
++
NHNH33
OO OO
+ HPO+ HPO44
2–2–
OOCCHCCH22CHCH22CHCOCHCO––
++
NHNH33
OO OO
++ NNHH33
PP
OO
––
OO
––
OO

Phosphodiesters
• A phosphodiester linkage between two
nucleotides is analogous to a peptide bond
between two amino acids.
• Two nucleotides joined by a phosphodiester
linkage gives a dinucleotide.
Three nucleotides joined by two
phosphodiester linkages gives a trinucleotide, etc.
(See next slide)
• A polynucleotide of about 50 or fewer
nucleotides is called an oligonucleotide.

Fig. 28.1
The
trinucleotide
ATG
• phosphodiest
er linkages
between 3' of
one
nucleotide
and 5' of the
next
3'
5'
HOCH2
O
O
N
N
N
N
P OCH2O
HO
NH2
OCH2
O
NH
N
N
N
HO
O
NH2
HO
O P
H3C
O
O
O
O
NH
N
AA
TT
GG
free 5' endfree 5' end
free 3' endfree 3' end

Nucleic Acids
• Nucleic acids first isolated in 1869 (Johann
Miescher)
• Oswald Avery discovered (1945) that a substance
which caused a change in the genetically
transmitted characteristics of a bacterium was
DNA.
• Scientists revised their opinion of the function of
DNA and began to suspect it was the major
functional component of genes.

Composition of DNA
• Erwin Chargaff (Columbia Univ.) studied DNAs
from various sources and analyzed the
distribution of purines and pyrimidines in them.
• The distribution of the bases adenine (A), guanine
(G), thymine (T), and cytosine (C) varied among
species.
• But the total purines (A and G) and the total
pyrimidines (T and C) were always equal.
• Moreover: %A = %T, and %G = %C

Composition of Human DNA
• Adenine (A) 30.3% Thymine (T) 30.3%
• Guanine (G) 19.5% Cytosine (C) 19.9%
• Total purines: 49.8% Total pyrimidines: 50.1%
For example:For example:
PurinePurine PyrimidinePyrimidine

Structure of DNA
• James D. Watson and Francis H. C. Crick proposed
a structure for DNA in 1953.
• Watson and Crick's structure was based on:
•Chargaff's observations
•X-ray crystallographic data of Maurice
Wilkins and Rosalind Franklin
•Model building

Base Pairing
• Watson and Crick proposed that A and T were
present in equal amounts in DNA because of
complementary hydrogen bonding.
2-deoxyribose2-deoxyribose N
NN
N N
H
H
N
N
O CH3
O
H
2-deoxyribose2-deoxyribose
AA TT

Base Pairing
• Watson and Crick proposed that A and T were
present in equal amounts in DNA because of
complementary hydrogen bonding.

Base Pairing
• Likewise, the amounts of G and C in DNA were
equal because of complementary hydrogen
bonding.
N
NN
N O
N
H
H
H
N
N
N
O
H
H
GG CC

Base Pairing
• Likewise, the amounts of G and C in DNA were
equal because of complementary hydrogen
bonding.

The DNA Duplex
• Watson and Crick proposed a double-stranded
structure for DNA in which a purine or pyrimidine
base in one chain is hydrogen bonded to its
complement in the other.
• •Gives proper Chargaff ratios (A=T and G=C)
• •Because each pair contains one purine and
one pyrimidine, the A---T and G---C distances
between strands are approximately equal.
• •Complementarity between strands suggests
a mechanism for copying genetic information.

Fig. 28.4
• Two antiparallel
strands of DNA
are paired by
hydrogen bonds
between purine
and pyrimidine
bases.
O
O
Ğ
O
Ğ
O
Ğ
O
O
O
O
O
OP
O
O
P O
O
O
P O
O
O
O
O
O
Ğ
O
Ğ
O
Ğ
O
O
O
O
O P
O
O
PO
O
O
PO
O
O
C G
AT
AT
CG
3'
5'
5'
5'
5' 5'
5'
5'
5'
3'
3'
3'
3'
3'
3'
3'

Fig. 28.5
• Helical structure
of DNA. The
purine and
pyrimidine bases
are on the inside,
sugars and
phosphates on
the outside.

DNA is coiled
• A strand of DNA is too long (about 3 cm in length)
to fit inside a cell unless it is coiled.
• Random coiling would reduce accessibility to
critical regions.
• Efficient coiling of DNA is accomplished with the
aid of proteins called histones.

Histones
• Histones are proteins rich in basic amino acids
such as lysine and arginine.
• Histones are positively charged at biological pH.
DNA is negatively charged.
• DNA winds around histone proteins to form
nucleosomes.

Histones
Each nucleosome contains one and three-quartersEach nucleosome contains one and three-quarters
turns of coil = 146 base pairs.turns of coil = 146 base pairs.
Linker contains about 50 base pairs.Linker contains about 50 base pairs.

Histones
NucleosomeNucleosome == Histone proteinsHistone proteins + Supercoiled DNA+ Supercoiled DNA

Fig. 28.8 DNA Replication
• The DNA to be copied is a double helix, shown here as flat
for clarity.
A T C C G T A G G A T T A GC
5'3'
AT G CG A T C C T G A A T C
3'5'
The two strands begin to unwind. (next slide)

• Each strand will become a template for construction of its
complement.
A T C C G T A G
G
A
T
T A
G
C
AT G CG A
T
C
C T
G
A
A
T C
3'
5'
5'
3'

• Two new strands form as nucleotides that are
complementary to those of the original strands are joined
by phosphodiester linkages.
A'
T'
G'
C'
A T C C G T
A G
G A
T
T A G
C
5'
3'
AT G CG A
T
C
C T
G
A
A
T C
5'
5'
T' A'
3'
3'
leading strand
lagging strand
3'
5'
C'T'
A'A'
Polynucleotide chains grow in
the 5'-3' direction—continuous in
the leading strand, discontinuous
in the lagging strand.

• Two duplex DNAs result, each of which is identical to the
original DNA.
A' T' C' C' G' T' A' G' G' A' T' T' A' G'C'
5'3'
AT G CG A T C C T G A A T C
3'5'
A T C C G T A G G A T T A GC
5'3'
A'T' G' C'G' A' T' C' C' T' G' A' A' T' C'
3'5'
+

Elongation of the growing DNA chain
• The free 3'-OH group of the growing DNA chain
reacts with the 5'-triphosphate of the appropriate
nucleotide.

Fig. 28.9 Chain elongation
OHOH
CHCH22
OO
OO PP
OO
O–O–
OO PP
OO
O–O–
OO PP
OO
O–O–
OO––Adenine,
Guanine,
Cytosine, or
Thymine
••••
OHOH
CHCH22OPOOPO
OO
Adenine,
Guanine,
Cytosine, or
Thymine
OO
O–O–
••••
Poly-Poly-
nucleotidenucleotide
chainchain
Poly-Poly-
chainchain

Fig. 28.9 Chain elongation
OHOH
CHCH22
OO
OO PP
OO––
Adenine,
Guanine,
Cytosine, or
Thymine
OO
CHCH22OPOOPO
OO
Adenine,
Guanine,
Cytosine, or
Thymine
OO
O–O–
•••• ••••
Poly-Poly-
chainchain
Poly-Poly-
chainchain
OO
OO PP
OO
O–O–
OO PP
OO
O–O–
OO––––

DNA and Protein Biosynthesis
• According to Crick, the "central dogma" of
molecular biology is:
"DNA makes RNA makes protein."
• Three kinds of RNA are involved.
messenger RNA (mRNA)
transfer RNA (tRNA)
ribosomal RNA (rRNA)
• There are two main stages.
transcription
translation

Transcription
• In transcription, a strand of DNA acts as a
template upon which a complementary RNA is
biosynthesized.
• This complementary RNA is messenger RNA
(mRNA).
• Mechanism of transcription resembles mechanism
of DNA replication.
• Transcription begins at the 5' end of DNA and is
catalyzed by the enzyme RNA polymerase.

Fig. 28.10 Transcription
Only a section of about 10 base pairs in the DNAOnly a section of about 10 base pairs in the DNA
is unwound at a time. Nucleotides complementaryis unwound at a time. Nucleotides complementary
to the DNA are added to form mRNA.to the DNA are added to form mRNA.

The Genetic Code
• The nucleotide sequence of mRNA codes for the
different amino acids found in proteins.
• There are three nucleotides per codon.
• There are 64 possible combinations of A, U, G, and
C.
• The genetic code is redundant. Some proteins are
coded for by more than one codon.

Table 28.3 (p 1175)
• UUU Phe UCU Ser UAU Tyr UGU Cys U
• UUC Phe UCC Ser UAC Tyr UGC Cys C
• UUA Leu UCA Ser UAA Stop UGA Stop A
• UUG Leu UCG Ser UAG Stop UCG Trp G
• U
• C
• A
• G
• U
• C
• A
• G
• U
• C
• A
• G
UU CC AA GG
UU
CC
AA
GG
First letterFirst letter
Second letterSecond letter
Third letterThird letter

• UUU Phe UCU Ser UAU Tyr UGU Cys U
• UUC Phe UCC Ser UAC Tyr UGC Cys C
• UUA Leu UCA Ser UAA Stop UGA Stop A
• UUG Leu UCG Ser UAG Stop UCG Trp G
• CUU Leu CCU Pro CAU His CGU Arg U
• CUC Leu CCC Pro CAC His CGC Arg C
• CUA Leu CCA Pro CAA Gln CGA Arg A
• CUG Leu CCG Pro CAG Gln CCG Arg G
• AUU Ile ACU Thr AAU Asn AGU Ser U
• AUC Ile ACC Thr AAC Asn AGC Ser C
• AUA Ile ACA Thr AAA Lys AGA Arg A
• AUG Met ACG Thr AAG Lys ACG Arg G
• GUU Val GCU Ala GAU Asp GGU Gly U
• GUC Val GCC Ala GAC Asp GGC Gly C
• GUA Val GCA Ala GAA Glu GGA Gly A
• GUG Val GCG Ala GAG Glu GCG Gly G
UU CC AA GG
UU
CC
AA
GG

• U
• C
• UAA Stop UGA Stop A
• UAG Stop G
• U
• C
• A
• G
• AUU Ile ACU Thr AAU Asn AGU Ser U
• AUC Ile ACC Thr AAC Asn AGC Ser C
• AUA Ile ACA Thr AAA Lys AGA Arg A
• AUG Met ACG Thr AAG Lys ACG Arg G
• U
• C
• A
• G
UU CC AA GG
UU
CC
AA
GG
AUG is the "start" codon. Biosynthesis of all
proteins begins with methionine as the first amino
acid. This methionine is eventually removed after
protein synthesis is complete.
UAA, UGA, and UAG
are "stop" codons that
signal the end of the
polypeptide chain.

Transfer tRNA
• There are 20 different tRNAs, one for each amino
acid.
• Each tRNA is single stranded with a CCA triplet at
its 3' end.
• A particular amino acid is attached to the tRNA by
an ester linkage involving the carboxyl group of
the amino acid and the 3' oxygen of the tRNA.

Transfer RNA
• Example—Phenylalanine transfer RNA
One of the mRNA codons for phenylalanine is:One of the mRNA codons for phenylalanine is:
UUCUUC5'5' 3'3'
AAGAAG3'3' 5'5'
The complementary sequence in tRNA is calledThe complementary sequence in tRNA is called
thethe anticodonanticodon..

Fig. 28.11 Phenylalanine tRNA
OCCHCHOCCHCH22CC66HH55
++
NHNH33
OO
Anticodon
3'
5'
3'
5'

Ribosomal RNA
• Most of the RNA in a cell is ribosomal RNA
• Ribosomes are the site of protein synthesis. They
are where translation of the mRNA sequence to
an amino acid sequence occurs.
• Ribosomes are about two-thirds RNA and one-
third protein.
• It is believed that the ribosomal RNA acts as a
catalyst—a ribozyme.

Protein Biosynthesis
• During translation the protein is synthesized
beginning at its N-terminus.
• mRNA is read in its 5'-3' direction
begins at the start codon AUG
ends at stop codon (UAA, UAG, or UGA)

Fig. 28.12 Translation
• Reaction that occurs is
nucleophilic acyl
substitution. Ester is
converted to amide.
Methionine at N-terminusMethionine at N-terminus
is present as its N-formylis present as its N-formyl
derivative.derivative.

• Ester at 3' end of
alanine tRNA is Met-Ala.
• Process continues along
mRNA until stop codon
is reached.

AIDS
• Acquired immune deficiency syndrome
• More than 22 million people have died from AIDS
since disease discovered in 1980s
• Now fourth leading cause of death worldwide and
leading cause of death in Africa (World Health
Organization)

HIV
• Virus responsible for AIDS in people is HIV (human
immunodeficiency virus)
• Several strains of HIV designated HIV-1, HIV-2, etc.
• HIV is a retrovirus. Genetic material is RNA, not
DNA.

HIV
• HIV inserts its own RNA and an enzyme (reverse
transcriptase) in T4 lymphocyte cell of host.
• Reverse transcriptase catalyzes the formation of
DNA complementary to the HIV RNA.
• HIV reproduces and eventually infects other T4
lympocytes.
• Ability of T4 cells to reproduce decreases,
interfering with bodies ability to fight infection.

AIDS Drugs
• AZT and ddI are two drugs used against AIDS that
delay onset of symptoms.
O
NN OO
NN33
OO
HH33CC
HOCHHOCH22
OO
NNHH
AZTAZT
O
NN
OO
HOCHHOCH22
OO
NNHH
ddIddI
NN
NN
HH
HH HH
HH

AIDS Drugs
• Protease inhibitors are used in conjunction with
other AIDS drugs.
• Several HIV proteins are present in the same
polypeptide chain and must be separated from
each other in order to act.
• Protease inhibitors prevent formation of HIV
proteins by preventing hydrolysis of polypeptide
that incorporates them.

DNA Sequencing
• Restriction enzymes cleave the polynucleotide to
smaller fragments.
• These smaller fragments (100-200 base pairs) are
sequenced.
• The two strands are separated.

DNA Sequencing
• Single stranded DNA divided in four portions.
• Each tube contains adenosine, thymidine,
guanosine, and cytidine plus the triphosphates of
their 2'-deoxy analogs.
POCHPOCH22
OHOH
OOOO
OO
OHOH
PP
OO
OHOH
PP
OO
HOHO basebase
HHHHOO
OO

DNA Sequencing
• The first tube also contains the 2,'3'-dideoxy analog of
adenosine triphosphate (ddATP); the second tube the
2,'3'-dideoxy analog of thymidine triphosphate (ddTTP),
the third contains ddGTP, and the fourth ddCTP.
POCHPOCH22
OHOH
OOOO
OO
OHOH
PP
OO
OHOH
PP
OO
HOHO basebase
HHHH
OO

DNA Sequencing
• Each tube also contains a "primer," a short section
of the complementary DNA strand, labeled with
radioactive phosphorus (32
P).
• DNA synthesis takes place, producing a
complementary strand of the DNA strand used as
a template.
• DNA synthesis stops when a dideoxynucleotide is
incorporated into the growing chain.

DNA Sequencing
• The contents of each tube are separated by
electrophoresis and analyzed by autoradiography.
• There are four lanes on the electrophoresis gel.
• Each DNA fragment will be one nucleotide longer
than the previous one.

Figure 27.29
TT
TTGG
TTGGAA
TTGGAACC
TTGGAACCAA
TTGGAACCAATT
TTGGAACCAATTAA
TTGGAACCAATTAACC
TTGGAACCAATTAACCGG
TTGGAACCAATTAACCGGTT
ddAddA ddTddT ddGddG ddCddC
Sequence ofSequence of
fragmentfragment

Figure 27.29
TT
TTGG
TTGGAA
TTGGAACC
TTGGAACCAA
TTGGAACCAATT
TTGGAACCAATTAA
TTGGAACCAATTAACC
TTGGAACCAATTAACCGG
TTGGAACCAATTAACCGGTT
ddAddA ddTddT ddGddG ddCddC
AA
AACC
AACCTT
AACCTTGG
AACCTTGGTT
AACCTTGGTTAA
AACCTTGGTTAATT
AACCTTGGTTAATTGG
AACCTTGGTTAATTGGCC
AACCTTGGTTAATTGGCCAA
fragmentfragment
original DNAoriginal DNA

Human Genome Project
• In 1988 National Research Council (NRC)
recommended that the U.S. undertake the
mapping and sequencing of the human genome.
• International Human Genome Sequencing
Consortium (led by U.S. NIH) and Celera Genomics
undertook project. Orginally competitors, they
agreed to coordinate efforts and published draft
sequences in 2001.

DNA Profiling
• DNA sequencing involves determining the nucleotide
sequence in DNA.
• The nucleotide sequence in regions of DNA that code
for proteins varies little from one individual to
another, because the proteins are the same.
• Most of the nucleotides in DNA are in "noncoding"
regions and vary significantly among individuals.
• Enzymatic cleavage of DNA give a mixture of
polynucleotides that can be separated by
electrophoresis to give a "profile" characteristic of a
single individual.Dr. Wolf's CHM 424 28- 83

PCR
• When a sample of DNA is too small to be
sequenced or profiled, the polymerase chain
reaction (PCR) is used to make copies ("amplify")
portions of it.
• PCR amplifies DNA by repetitive cycles of the
following steps.
• 1. Denaturation
2. Annealing ("priming")
3. Synthesis ("extension" or "elongation")

Target regionTarget region
((aa) Consider double-stranded DNA containing) Consider double-stranded DNA containing
a polynucleotide sequence (the target region)a polynucleotide sequence (the target region)
that you wish to amplify.that you wish to amplify.
((bb) Heating the DNA to about 95°C causes the) Heating the DNA to about 95°C causes the
strands to separate. This is the denaturationstrands to separate. This is the denaturation
step.step.

((bb) Heating the DNA to about 95°C causes the) Heating the DNA to about 95°C causes the
strands to separate. This is the denaturationstrands to separate. This is the denaturation
step.step.
((cc) Cooling the sample to ~60°C causes one) Cooling the sample to ~60°C causes one
primer oligonucleotide to bind to one strand andprimer oligonucleotide to bind to one strand and
the other primer to the other strand. This is thethe other primer to the other strand. This is the
annealing step.annealing step.

((cc) Cooling the sample to ~60°C causes one) Cooling the sample to ~60°C causes one
primer oligonucleotide to bind to one strand andprimer oligonucleotide to bind to one strand and
the other primer to the other strand. This is thethe other primer to the other strand. This is the
annealing step.annealing step.
((dd) In the presence of four DNA nucleotides and) In the presence of four DNA nucleotides and
the enzyme DNA polymerase, the primer isthe enzyme DNA polymerase, the primer is
extended in its 3' direction. This is the synthesisextended in its 3' direction. This is the synthesis
step and is carried out at 72°C.step and is carried out at 72°C.

This completes one cycle of PCR.This completes one cycle of PCR.
((dd) In the presence of four DNA nucleotides and) In the presence of four DNA nucleotides and
the enzyme DNA polymerase, the primer isthe enzyme DNA polymerase, the primer is
extended in its 3' direction. This is the synthesisextended in its 3' direction. This is the synthesis
step and is carried out at 72°C.step and is carried out at 72°C.

This completes one cycle of PCR.This completes one cycle of PCR.
((ee) The next cycle begins with the denaturation) The next cycle begins with the denaturation
of the two DNA molecules shown. Both areof the two DNA molecules shown. Both are
then primed as before.then primed as before.

((ee) The next cycle begins with the denaturation) The next cycle begins with the denaturation
of the two DNA molecules shown. Both areof the two DNA molecules shown. Both are
then primed as before.then primed as before.
((ff) Elongation of the primed fragments completes) Elongation of the primed fragments completes
the second PCR cycle.the second PCR cycle.

((ff) Elongation of the primed fragments completes) Elongation of the primed fragments completes
the second PCR cycle.the second PCR cycle.
((gg) Among the 8 DNAs formed in the second) Among the 8 DNAs formed in the second
cycle are two having the structure shown.cycle are two having the structure shown.

The two contain only the target region andThe two contain only the target region and
and are the ones that increase disproportionatelyand are the ones that increase disproportionately
in subsequent cycles.in subsequent cycles.
((gg) Among the 8 DNAs formed in the second) Among the 8 DNAs formed in the second
cycle are two having the structure shown.cycle are two having the structure shown.

CycleCycle Total DNAsTotal DNAs Contain only targetContain only target
0 (start)0 (start) 11 00
11 22 00
22 44 00
33 88 22
44 1616 88
55 3232 2222
1010 1,0241,024 1,0041,004
2020 1,048,5661,048,566 1,048,5261,048,526
3030 1,073,741,8241,073,741,824 1,073,741,7641,073,741,764

Nucleic acid

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