chapter 3 structures and functions of nucleic acids
TRANSCRIPT
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Chapter 3
Structures and Functions of Nucleic Acids
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Nucleic acid
A biopolymer composed of nucleotides linked in a linear sequential order through 3’,5’ phosphodiester bonds
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Classification of nucleic acid
• Ribonucleic acid (RNA) is composed of ribonucleotides.– in nuclei and cytoplasm – participate in the gene expression
• Deoxyribonucleic acid (DNA) is composed of deoxyribonucleotides.– 90% in nuclei and the rest in mitochon
dria – store and carry genetic information; d
etermine the genotype of cells
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Interesting history
• 1944: proved DNA is genetic materials (Avery et al.)• 1953: discovered DNA double helix (Watson and Cri
ck)• 1968: decoded the genetic codes (Nirenberg)• 1975: discovered reverse transcriptase (Temin and
Baltimore)• 1981: invented DNA sequencing method (Gilbert and
Sanger)• 1985: invented PCR technique (Mullis)• 1987: launched the human genome project • 1994: HGP in China• 2001: accomplished the draft map of human genome
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Section 1
Chemical Components of Nucleic Acids
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nucleic acid nucleotidesphosphate
nucleosides
pentose
bases
§ 1.1 Molecular Constituents
Nucleic acid can be hydrolyzed into nucleotides by nucleases. The hydrolyzed nucleic acid has equal quantity of base, pentose and phosphate.
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N
N
NH
N1
23
4
567
89
N
N
NH
N
NH2
Adenine (A)
N
NH
NH
N
NH2
O
Guanine (G)
Base: Purine
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N
NH1
32
45
6
Base: Pyrimidine
Cytosine (C)
N
NH
NH2
O
Uracil (U)NH
NH
O
O
Thymine (T)
NH
NH
O
O
CH3
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Pentose
1´
2´3´4´
5´OH
O
CH2OH
OH OH
-D-ribose
OHO
CH2OH
OH
-D-2-deoxyribose
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Ribonucleoside
OHO
CH2
OHOH
N
N
NH2
O
1´
1
Purine N-9 or pyrimidine N-1 is connected to pentose (or deoxypentose) C-1’ through a glycosidic bond.
glycosidic bond
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P
O
O
OH
OHO
CH2
OHOH
N
N
NH2
O
A nucleoside (or deoxynucleoside) and a phosphoric acid are linked together through the 5’-phosphoester bond.
Ribonucleotide
phosphoester bond
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base nucleoside nucleotide
guanine guanosine guanosine monophosphate
(GMP)
cytosine cytidine cytidine monophosphate
(CMP)
adenine adenosine adenosine monophosphate
(AMP)
uracil uridine uridine monophosphate
(UMP)
(NMP)
Nomenclature
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base nucleoside nucleotideguanine deoxyguanosine deoxyguanosine monophosphate
(dGMP)
cytosine deoxycytidine deoxycytidine monophosphate
(dCMP)
adenine deoxyadenosine deoxyadenosine monophosphate
(dAMP)
thymine deoxythymidine deoxythymidine monophosphate
(dTMP)
(dNMP)
Nomenclature
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Nucleic acid
base ribose
DNA A、 G、 C、 T deoxyribose
RNA A、 G、 C、 U ribose
Composition of DNA and RNA
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Nucleic acid derivatives
Multiple phosphate nucleotides adenosine monophosphate (AMP)adenosine diphosphate (ADP)adenosine triphosphate (ATP)
NO
CH2O
OHOH
N
NN
NH2
P
O
OH
OH
AMPAMP
NO
CH2O
OHOH
N
NN
NH2
P
O
OH
OP
O
OH
OH
ADPADP
NO
CH2O
OHOH
N
NN
NH2
P
O
OH
OP
O
OH
OP
O
OH
OH
ATPATP
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Cyclic ribonucleotide: 3’,5’-cAMP, 3’,5’-cGMP, used in signal transduction
NO
CH2O
OHO
N
NN
NH2
PO
OH
cAMPcAMP
Nucleic acid derivatives
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Biologically active systems containing ribonucleotide: NAD+, NADP+, CoA-SH
Nucleic acid derivatives
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The -P atom of the triphosphate group of a dNTP attacks the C-3’ OH group of a nucleotide or an existing DNA chain, and forms a 3’-phosphoester bond.
Phosphoester bond formation
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Nucleic acid chain extension
A nucleic acid chain, having a phosphate group at 5’ end and a -OH group at 3’ end, can only be extended from the 3’ end.
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Phosphodiester bonds
Alternative phosphodiester bonds and pentoses constitute the 5’-3’ backbone of nucleic acids.
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Section 2
Structures and Functions of Nucleic Acids
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§ 2.1 Primary Structure
• The primary structure of DNA and RNA is defined as the nucleotide sequence in the 5’ – 3’ direction.
• Since the difference among nucleotides is the bases, the primary structure of DNA and RNA is actually the base sequence.
• The nucleotide chain can be as long as thousands and even more, so that the base sequence variations create phenomenal genetic information.
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P P
A
P
C
P
C
P
T
P
G
OH
C
P
T
P
A
P
A
5' 3'
pApCpTpGpCpTpApApC-OH 3'
5' ACTGCTAAC 3'
5'
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§ 2.2 Secondary structure
The secondary structure is defined as the relative spatial position of all the atoms of nucleotide residues.
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§ 2.2.a Chargaff’s rules
• The base composition of DNA generally varies from one species to another.
• DNA isolated from different tissues of the same species have the same base composition.
• The base composition of DNA in a given species does not change with its age, nutritional state, and environmental variations.
• The molarity of A equals to that of T, and the molarity of G is equal to that of C.
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Molarity of bases
A G C T A/T G/C G+C Pu/Py
E. coli 26.0 24.9 25.2 23.9 1.09 0.99 50.1 1.04
Tuberculosis
15.1 34.9 35.4 14.6 1.03 0.99 70.3 1.00
Yeast 31.7 18.3 17.4 32.6 0.97 1.05 35.7 1.00
Cow 29.0 21.2 21.2 28.7 1.01 1.00 42.4 1.01
Pig 29.8 20.7 20.7 29.1 1.02 1.00 41.4 1.01
Human 30.4 19.9 19.9 30.1 1.01 1.00 39.8 1.01
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Historic X-ray diffraction picture
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Building a milestone of life
James Watson and Francis Crick proposed a double helix model of DNA in 1953.
It symbolized the new era of modern biology.
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• Two DNA strands coil together around the same axis to form a right-handed double helix (also called duplex).
• The two strands run in opposite directions, i.e., antiparallel.
• There are 10 base pairs or 3.4nm per turn and the diameter of the helix is 2.0nm.
§ 2.2.b Double helix of DNA
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Antiparallel
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The hydrophilic backbone is on the outside of the duplex, and the bases lie in the inner portion of the duplex.
Backbone and bases
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• The two strands of DNA are stabilized by the base interactions.
• The bases on one strand are paired with the complementary bases on another strand through H-bonds, namely G≡C and A=T.
• The paired bases are nearly planar and perpendicular to helical axis.
• Two adjacent base pairs have base-stacking interactions to further enhance the stability of the duplex.
Base interactions
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Watson-Crick base pair
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Watson-Crick base pair
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Base-stacking interaction
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Major and minor grooves
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Groove binding
Small molecules like drugs bind in the minor groove, whereas particular protein motifs can interact with the major grooves.
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§ 2.2.c Polymorphisms of DNA
• DNA can resume different forms depending upon their chemical microenvironment, such as ionic strength and relative humidity.
• B-form DNA is the predominant structure in the aqueous environment of the cells.
• A-form and Z-form are also native structures found in biological systems.
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Feature A-DNA B-DNA Z-DNA
Helix rotation Right-handed Right-handed Left-handed
Base pair per turn 11 10 12
Pitch 2.46nm 3.4nm 4.56nm
Helical diameter 2.55nm 2.0nm 1.84nm
Rise per base pair 0.26nm 0.34nm 0.37nm
Glycosyl formation Anti- Anti- Anti- at C, syn- at G
Rotation between adjacent base pair
33º 36º -60º per dimer
Relative humidity 75% 92%
Structural features of DNAs
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Triplet DNA
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Hoogsteen base pair
The third strand is using Hoogsteen H-bonds to pair with bases on the first strand.
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G-quartet DNA
• The telomere of DNA is a G-righ sequence, such as
5’ (TTGGGG)n 3’
• 4 G residues constitute a plane which is stabilized by Hoogsteen H-bonds.
G
G
T
T
T
T
TT
G
T G
T
T
G
5'3'
T
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G-quartet of DNA
Four strands are arranged in either parallel or antiparallel manner.
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§ 2.3 Supercoil Structure
• The two termini of a linear DNA could be joined to form a circular DNA.
• The circular DNA is supercoiled, and supercoil can be either positive or negative.
• Only the supercoiled DNA demonstrate biological activities.
§ 2.3.a Supercoil structure
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EM image of supercoiled DNA
Circular DNAs in nature, in general, are negatively supercoiled.
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§ 2.3.b Prokaryotic DNA
• Most prokaryotic DNAs are supercoiled.
• Different regions have different degrees of supercoiled structures.
• About 200 nts will have a supercoil on average.
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§ 2.3.c Eukaryotic DNA
• DNA in eukaryotic cells is highly packed.
• DNA appears in a highly ordered form called chromosomes during metaphase, whereas shows a relatively loose form of chromatin in other phases.
• The basic unit of chromatin is nucleosome.
• Nucleosomes are composed of DNA and histone proteins.
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Nucleosome
• DNA: ~ 200 bps
• Histone: basic proteins with many Lys and Arg residues– H2A (x2), – H2B (x2), – H3 (x2), – H4 (x2)
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Beads on a string
• 146 bp of negatively supercoiled DNA winds 1 ¾ turns around a histone octomer.
• H1 histone binds to the DNA spacer.
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The total length of 46 human chromosomes is about 1.7 m, and becomes 200 nm long after 5 times condensation.
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§ 2.4 Functions of DNA
DNA is fundamental to individual life in terms of
• They are the material basis of life inheritance, providing the template for RNA synthesis.
• They are the information basis for biological actions, carrying the genetic information.
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• DNA is able to replicate itself in a high fidelity to ensure the genetic information transfer from one generation to the next.
• DNA can be used as a template to synthesize RNA (transcription), and RNA is further used as the template to synthesize proteins (translation).
• DNA posses the inherent and the mutant properties to create the diversity and the uniformity of the biological world.
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• A gene is defined as a DNA segment that encodes the genetic information required to produce functional biological products.
• A gene includes coding regions as well as non-coding regions.
• Genome is a complete set of genes of a given species.
Gene and genome
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Section 3
Structures and Functions of RNA
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Classification
• mRNA (messenger RNA): template for protein synthesis
• tRNA (transfer RNA): AA carrier
• rRNA (ribosomal RNA): a component of ribosome for protein synthesis
• hnRNA (heterogeneous nuclear RNA): precursor of mRNA
• snRNA (small nuclei RNA): small RNAs for processing and transporting hnRNA
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Classes of eukaryotic RNAs
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Unique features
• RNA is single stranded, in general.
• RNA has self-complementary intrachain base paring.
• The double helical regions of RNA are of the A-form.
• RNA is susceptible to hydrolysis.
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§ 3.1 Messenger RNA
• mRNAs constitute 5% of total RNAs.
• mRNAs vary significantly in life spans.
• hnRNA is the precursor of mRNA.
mRNA is the template for protein synthesis, that is, to translate each genetic codon on mRNA into each AA in proteins. Each genetic codon is a set of three continuous nucleotides on mRNA.
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AUG AAA.....AAA
5' non-coding region 3' non-coding region
coding region
5'-cap 3'-poly A tail
UAA
mRNA structure
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• hnRNA contains introns and exons.
• Exons are the sequences encoding proteins, and introns are non-coding portions.
• Splicing process of hnRNA removes introns and makes mRNA become matured.
• The matured mRNA has special structure features, including 5’-cap and 3’-poly A tail.
mRNA maturation
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5’-cap
O
N
NN
N
NH2
O
OCH3O
HHH
CH2
H
OP
O-
O
O
HN
N
N
O
H2N N O
OH
H H
H
CH2
HOH
O PO
O-
CH3
P
O-
O5'
2'3'
5'
mRNA chain
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5’-cap addition
ppp5'NpNp
pp5'NpNp
GTP
PPi
G5'ppp5'NpNp
methylating at G7
methylating at C2' of the first andsecond nucleotides after G
forming 5'-5' triphosphategroup
removing phosphate group
m7GpppNpNp
m7Gpppm2'Npm2'Np
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5’-cap addition
• Methylation can occur at different sites on G or A.
• 5’-cap can be bound with CBP, benefiting transporting from nucleus to cytoplasm.
• 5’-cap can be recognized by translation initiation factor.
• It protects the 5’-end from exonucleases.
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Poly A tail
• 20-200 adenine nucleotides at 3’ end
• a un-translated sequence.
• Related with mRNA degradation that begins with poly A tail shortening.
• Associate with poly A tail binding proteins for protection
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Poly A tailing
5'
(Y=pyrimidine)
AAUAAA (~20nt) YA UUGUGUGUUG 3'
5' AAUAAA (~20nt) Y
5' AAUAAA (~20nt) YAAAAAAAAA AAAAAA
signal sequencefor poly A tail GU-rich region
~250nt
5' AAUAAA (~20nt) YAAAAAAAAA
poly A binding protein
AAAAAA
cleavage site
3'
3'
ATP
PPi
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hnRNA
mRNA
hnRNA splicing
intron exon
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AUG AAA.....AAA
5' non-coding region 3' non-coding region
coding region
5'-cap 3'-poly A tail
UAA
Matured mRNA of eukaryote
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§ 3.2 Transfer RNA
• tRNA is about 15% of total RNA.
• tRNA is 65-100 nucleotides long.
• There are at least 20 types of tRNA in one cell.
tRNA serves as an amino acid carrier to transport AA for protein synthesis.
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• The overall structure is a cloveleaf, reversed L-shape structure.
• There are three loops (DHU loop, anticodon loop, TψC loop), and four stems.
• The 3-D structure is stabilized by hydrogen bonds of local intrachain base pairs on these stems.
Structure of tRNA
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Reversed L-shape structure
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• A tRNA molecule has an amino acid attachment site and a template-recognition site, bridging DNA and protein.
• The template-recognition site is a sequence of three bases called the anticodon complementary to the mRNA codon.
Two key sites of tRNA
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Codon and anticodon
The anticodon on tRNA pairs with the codon on mRNA.
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Amino acid attachment
• The OH group at the 3' end of tRNA links covalently to an amino acid.
• Only the attached AA becomes activated and capable of being transported.
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Rare Bases
tRNA contains a high portion of unusual bases.
NH
N
H2C
H2C
O
O
R
Dihydrouridine(DHU)
NHHN
O
O
R
pseudouridine()
NH
NN
N
O
NH2
R
CH3
7-Methylguanosine(mG)
1
35
5
3 1
3
5 17
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• rRNA is the most abundant RNA in cells (>80%).
• rRNA assembles with numerous ribosomal proteins to form ribosomes.
§ 3.3 Ribosomal RNA
rRNA provides a proper place for protein synthesis.
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• Ribosomes associate with mRNA to form a place for protein synthesis.
• Ribosomes of eukaryotes and prokaryotes are similar in shapes and functions.
Ribosomes
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Components of ribosomes
Prokaryote Eukaryote
(E.coli) (Liver of mouse)
Smaller subunit 30s 40s
rRNA 16s 1542 nucleotides 18s 1874 nucleotides
proteins 21 40% of total weight 33 50% of total weight
Larger subunit 50s 60s
rRNA 23s 2940 nucleotides 28s 4718 nucleotides
5s 120 nucleotides 5.85s 160nucleotides
5s 120nucleotides
proteins 31 30% of total weight 49 35% of total weight
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Ribosome of E. coli
70S ribosome
50S large subunit
23S rRNA 5S rRNA
31 proteins
16S rRNA
21 proteins30S small subunit
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Secondary structure of 18S rRNA
The secondary structure of rRNA has many loops and stems, which can bind ribosomal proteins to form an assembly for protein synthesis.
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m7GpppG AAA...AAA
核糖体大亚基
核糖体小亚基
E位
P位
A位
mRNA
氨基酸
核糖体移动方向
进位退位
N末端
肽链
Ribosomal complex
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核糖体
合成中的多肽
5'末端
3'末端
mRNA
Polysomes
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EM of polysomes
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Section 4
Physical and Chemical Properties of Nucleic Acids
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General properties
• Acidity– Negative backbone
• Viscosity– Concentration and aggregation effects
• Optical absorption– UV absorption due to aromatic groups
• Thermal stability– Disassociation of dsDNA (double-stranded DN
A) into two ssDNAs (single-stranded DNA)
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§ 4.1 UV Absorption
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Quantify DNAs or RNAs
OD260=1.0 equals to
50μg/ml dsDNA
40μg/ml ssDNA (or RNA)20μg/ml oligonucleotide
Determine the purity of nucleic acid samples
pure DNA: OD260/OD280 = 1.8
pure RNA: OD260/OD280 = 2.0
Application of OD260
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Transition of dsDNA to ssDNA
The absorbance at 260nm of a DNA solution increases when a dsDNA is melted into two single strands. The change is called hyperchromicity.
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Melting curve of dsDNA
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DNA melting
• Melting curve: a graphic presentation of the absorbance of dsDNA at 260nm versus the temperature.
• Melting temperature (Tm): the temperature at which the UV adsorption reaches the half of the maximum value, also means that about 50% of the dsDNA is disassociated into the single-stranded DNA.
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Melting curve shift
Tm of dsDNA depends on its average G+C content. The higher the G+C content, the higher the Tm.
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§ 4.2 Thermal stability
• Dissociation of dsDNA into two ssDNAs is referred to as denaturation.
• Denaturation can be partially and completely.
• The nature of the denaturation is the breakage of H-bonds.
• Denaturation is a common and important process in nature.
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Cooperative unwinding of DNA strands
Extremes in pH orhigh temperature
Denaturation of DNA
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EM image of denatured DNA
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Renaturation of DNA
Two separated complementary DNA strands can rejoin together to form a double helical form spontaneously when the temperature or pH returns to the biological range. This process is called renaturation or annealing.
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§ 4.3 Hybridization
• The ability of DNA to melt and anneal reversibly is extremely important.
• An association between two different polynucleotide chains whose base sequences are complementary is referred to as hybridization.
• The stability of the hybridized strand depends on the complementary degree.
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Two dsDNA molecules from different species are completely denutured by heating. When mixed and slowly cooled, complementary DNA strands of each species will associate and anneal to form normal duplexes.
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• Two ssDNAs, two ssRNAs, as well as one ssDNA and one ssRNA can also be hybridized.
• Ionic strength, degree of complementary, temperature, as well as base composition, fragment length of nucleic acids will affect the hybridization.
• It is a common phenomenon in biology, and has been used as a convenient techniques in medicine and biology.
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• complementary hybridization
probe: …. TAGCTGAG …target: …. ATCGACTC …
probe: …. TAGCTGAG …non-target: …. ATCAGCTC …
• mismatched hybridization
Target DNA detection
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Applications
• Gene structure and expression
• Microarray or gene chip
• mRNA separation
• Gene diagnosis and therapy
• PCR technique
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Section 5
Nuclease
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Nucleases are enzymes that are able to hydrolyze phosphoester bonds and cleave DNA or RNA into fragments.
Definition and classification
• Deoxyribonuclease (DNase) - specially cleave DNA
Ribonuclease (RNase) - specially cleave RNA
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ExonucleasesThey can cleave terminal nucleotides either from 5’-end or from 3’-end, such as enzymes used in the DNA replication.
Endonucleases They can cleave internally at either 3’ or 5’ side of a phosphate group, such as the restriction endonucleases used to construct the recombinant DNA.
Classification
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5’
5’3’
3’
Endonuclease
Endonuclease
Exonuclease
Exonuclease
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Applications
• Participate in DNA synthesis and repair, as well as RNA post-translational modification
• Digest nucleic acids of food for better absorption
• Degrade the invaded nucleic acids
• Construct the recombinant DNA