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Bi4025en
Molecular Biology
Mgr. Jiří Kohoutek, Ph.D.
1    Department of Experimental Biology
Lecture 2
• Nucleic acids: primary, secondary and tertiary structure of nucleic acids, conformation of DNA and RNA, different conformations of DNA and their significance for biological systems, genetic information and genetic code.
2    Department of Experimental Biology
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Hunt for the structure of DNA
• Watson and Crick restricted themselves to what they saw as chemically and biologically reasonable in terms of DNA structure.
• A breakthrough occurred in 1952, when Erwin Chargaff visited Cambridge and inspired Crick with a description of experiments he had published in 1947.
• Chargaff had observed that the proportions of the four nucleotides vary between one DNA sample and the next, but that for particular pairs of nucleotides__, ■-
• guanine and cvtosine
• adenine and thymine
• the two nucleotides are always present in equal proportions.
3    Department of Experimental Biology
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Discovery of DNA structure
* 1953: James Watson a Francis Crick derived the structure of DNA on the basis of the following data:
* Chemical data: Erwin Chargaff principles:
• the concentration of thymine and adenine is the same
• the concentration of cytosine and guanine is the same.
* Physical data: Maurice Wilkins a Rosalind Franklin after exposure of purified DNA molecules to X-rays, there is a characteristic scattering of rays that signal method of arranging DNA components into a helix.
Department of Experimental Biology
Structure of DNA
Levene's
tetranucleotide theories
dGMP i z	dCMP
o 0=0.-0	O
o	" O--0=Q 0 ? 1 -b-0 —fT.
T dTMP °	z CJAMP
• Features   of   the   proposed DNA structure allow:
o encoding genetic information in the form of ordered bases (denial of Levene's tetranucleotide theories)
o replication of DNA molecule is based on complementary pairing of bases.
group
m «,    1   ,* d„„   ,-k   m   hm MUNI
5    Department of Experimental Biology https://sites.google.eom/a/kcsd.k12.or.us/nickersonl/dna-model-1
O U i.
Structure of DNA
Chemical properties of bases:
Spontaneous mutation of DNA bases can also occur by tautomerization:
-ex
cnol-kelo (automcrism
>Nv ^NH,
H
I
o enol / keto o amino / imino
nmintvminm lautoinensni
• Tautomerization is a net process by which protons are transferred from one site to another by a series of steps in which the solvent is an intermediary.
(A) Standard base pairing arrangements
Ck H
~t "Vi
o
-n
-n
Thymine (keto)     Adenine (amino]
(B) Anomalous base pairing arrangement
o-h-
* o-
Thymine (enol)
■h n
)-n
h
6    Department of Experimental Biology
Green Chemistry, An Inclusive Approach, 2018, Pages 109-128
Vh---------on
—h-m y\
Vi—(\ >=^
/     £---------h-n
h
Cytosine (amino)       Guanine (keta)
h
H
u
Guanine (keto)      Cytosine (imino)    Adenine (amino)
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Types of Nucleic acids - DNA
• DNA forms the genome of prokaryotes, eukaryotes and DNA-viruses.
• gDNA-genomic, mtDNA- mitochondrial, cpDNA-chloroplast, pDNA-plasmid, recDNA- recombinant, rDNA-ribosomal, aDNA-ancient.
• cDNA (copy DNA, complementary DNA).
• dsDNA- double-stranded , ssDNA- single-stranded, cccDNA- covalently closed circular, ocDNA- open circle, NnDNA- linear.
• A-DNA, B-DNA, Z-DNA- conformation influenced by sequence and environment.
• Special forms of DNA - C-DNA, D-DNA and E-DNA.
Department of Experimental Biology
June 1981 Plasmid 5(3):371-3
https://microbenotes.com/different-forms-of-dna-b-form-a-form-z-form/
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Types of Nucleic acids - RNA
• RNA - forms the genome of RNA-viruses, in cellular organisms it is a component of ribosomes and perform various functions in the transmission and realization of genetic information.
• mRNA- mediator, hnRNA- heteronuclear, tRNA - transfer, rRNA-ribosomal, tmRNA- transfer-messenger RNA
• snRNA - small-nuclear, snoRNA - small nucleolar, scRNA - small cytoplasmic, gRNA-guide, crRNA-CRISPR RNA
• miRNA, siRNA, shRNA, piRNA
• ribozyme: ribonucleic and/+ enzyme.
8    Department of Experimental Biology
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Nucleotide
Nucleotides
• phosphatic acid (PHOPHATE)
• pentose (SUGAR) o ribose
o deoxyribose
• organic base (BASE) o purine base adenine guanine
o pyrimidine base cytosine thymine uracil
9    Department of Experimental Biology
NUCLEOTIDES
A nucleotide consists of a nitrogen-containing base, a five-carbon sugar, and one or more phosphate groups.
BASE
Nil
PHOSPHATE
Nucleotides are the subunits of
the nucleic acids
OH OH SUGAR
Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015
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Sugar - Pentose in Nucleic acids
Sugar - Pentose
• ribose in ribonucleic acids (RNA)
• deoxyribose in deoxyribonucleic acid (DNA)
• the difference is in the presence or absence of hydroxyl groups on 2-carbon
SUGARS
HOCH2 OH
C 5'
PENTOSE
a five-carbon sugar
11
|i-D-ribose
used in ribonucleic acid
two kinds are used
oh oh
i ioc'i l2
OH
Each numbered carbon on the sugar of a nucleotide is followed by a prime mark; therefore, one speaks of the "5-prime carbon," etc.
[i-D-2-deoxyribose
used in deoxyribonucleic acid
10 Department of Experimental Biology
Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015
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p-N-glycosidic bond in Nucleic acids
A/-glycosidic bond
The base is linked to the same carbon (C1) used in sugar-sugar bonds.
11   Department of Experimental Biology
Sugar - Pentose
• bases are attaching to sugar by (3-N-glycosidic bond which is a nitrogen-carbon linkage between the 9' nitrogen of purine bases or 1' nitrogen of pyrimidine bases and the 1' carbon of the sugar group.
N
I
HC
H
2 6
CH CH
Pyrimidine
Purine
https://slideplayer.com/slide/7012073/
Alberts et al.: Molecular biology of the cell. Garland Sci. 2008, 2015
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Pyrimidine and Purine bases in Nucleic acids
Bases
• pyrimidine (cytosine, thymine, uracil)
Bases
• purine (adenine, guanine)
BASES
NH,
O
HC Nil
HC HC,
cytosine
uracil
,C
N I I
O
H3C
"NI i
thymine   N %o
H
The bases are nitrogen-containing ring compounds, either pyrimidines or purines.
'N
pyrimidine
.N
N
'n
n
purine
NH-,
adenine ,NSr/
HC' A
\   „c ^ch
N' i i
N
O
.C
•\JI i
# II
hc; II G
guanine   n \ <y
H N "NH2
12  Department of Experimental Biology
Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015
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Phosphates in Nucleic acids
PHOSPHATES
The phosphates are normally joined to the C5 hydroxyl of the ribose or deoxyribose sugar (designated 5'). Mono-, di-( and triphosphates are common.
O II
"O— P— O— CH2 I
as in AMP
O
O
TJ— P —O — P —O —CH,
as m AD P
Q-
O
Q-
O
O
O— P—O—P—O—P—O—CH-
cr
Cr
I
as in ATP
The phosphate makes a nucleotide negatively charged.
13 Department of Experimental Biology
Phosphates
• attached to 5' sugar carbon by ester bound,
• provides the nucleotides with negative charge.
base
ester bond
»-0-C-
phosphate
JA u      u/-r glycoaidic
\f    V bond
HO H
ribose
https://www.sparknotes.com/biology/molecular/structureofnucleicacids/section1/ Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015
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Phosphates in Nucleic acids
NH;
O
"O— P— O— CH2 I
O"
o
o
O"
o
er o
as in AMP
O— P —O— P —O—CH:
as in ADP
O
"O— P —O— P —O— P —O—CH2 I
o-     o- ey-
as in
ATP
0" !
O
N
n
O O"
II I
HO-P-0-P-O-P-O-CH
.....
O" o
N
>
N
OH OH
J
Adenosine 5'-monophosphate (AMP)
^-Y-'
Adenosine 5'-diphosphate (ADP)
_J
-Y-
Adenosine S'-triphosphate (ATP)
14 Department of Experimental Biology
https://basicmedicalkey.com/nucleotides/
Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015
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Nucleosides and Nucleotides in Nucleic acids
• Nucleotides are phosphorylated Nucleosides.
NOMENCLATURE
BASE	NUCLEOSIDE	ABBR.
adenine	adenosine	A
guanine	guanosine	G
cytosine	cytidine	C
uracil	uridine	u
thymine	thymidine	T
A nucleoside or nucleotide is named according to its nitrogenous base.
Single letter abbreviations are used variously as shorthand for (1) the base alone, (2) the nucleoside, or (3) the whole nucleotide—
the context will usually make clear which of the three entities is meant. When the context is not sufficient, we will add the terms "base", "nucleoside", "nucleotide", or—as in the examples below—use the full 3-letter nucleotide code.
AMP   - adenosine monophosphate dAMP = deoxyadenosine monophosphate UDP    = uridine diphosphate ATP    = adenosine triphosphate
BASE + SUGAR = NUCLEOSIDE
BASE + SUGAR + PHOSPHATE = NUCLEOTIDE
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15 Department of Experimental Biology Alberts et al.: Molecular biology of the cell. Garland Sci. 2008, 2015 _ _
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Nucleosides and Nucleotides in Nucleic acids
TABLE 8-1	Nucleotide and Nucleic Acid Nomenclature		
Base	Nucleoside	Nucleotide	Nucleic add
Purines			
Adenine	Adenosine	Adenylate	UNA
	Deoxyadenosine	Deoxyadenylate	DNA
Guanine	Guanosine	Guanylate	RNA
	Deoxyguanosine	Deoxyguanylate	DNA
Pyrimidines			
Cytosine	Cytidine	Cytidylate	RNA
	Oeoxycytidine	Deoxycytidylate	DNA
Thymine	Thymidine or deoxythymidine	Thymidylate or deoxythymidylate	DNA
Uracil	Uridine	Uridylate	RNA
Note: "Nucleoside" and "nucleotide" are generic terms that include both ri bo- and deoxyribo- forms. Also, ribonucleosides and ribonucleotides are here designated simply as nucleosides and nucleotides {e.g., riboadenosine as adenosine), and deoxyribonucleosides and deoxyribonucleotides as deoxynucleosides and deoxy nucleotides (e.g., deoxyriboadenosine as deoxyadenosine). Both forms of naming are acceptable, but the shortened names are more commonly used.Thymine is an exception;"ribothymidine" is used to describe its unusual occurrence in RNA.
Table 8-1
Lehninger Principles of Biochemistry, Fifth Edition
0 2008 W. H. Freeman and Company
16 Department of Experimental Biology
Lehningeret al.: Principles of Biochemistry, W. H. Freeman and Company, 2008
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Deoxy ribonucleotides
Nucleotide is composed of a base (adenine, guanine, cytosine, thymine) attached to a sugar (deoxyribose) to form a nucleoside.
The nucleoside with an attached phosphate group makes it nucleotide.
The name of the nucleoside containing the base adenine is deoxyadenosine and if the phosphate group is attached at the carbon numbered 5' (five prime) then the formal name of the nucleotide is 2'deoxyadenosine 5'-monophosphate (dAMP).
2'-Deoxyadenosine5'-monophosphate (Deoxyadenylate, dAMP)
OH H
2'-Deoxyguano$ine 5'-monophosphate (Deoxyguanylate, dGMP)
e 1
uO — P = 0
I
0
1
CH
H,C
OH H
2'-Deoxycytidine 5'-monophosphate (Deoxycytidylate, dCMP)
2'-Deoxythymidine S'-monophosphate (Thymidylate,dTMP)
Figure 19-9 Principles of Biochemistry, 4/e © 2006 Pearson Prentice Hall,Inc.
17 Department of Experimental Biology
https://courses.lumenlearning.com/microbiology/chapter/structure-and-function-of-dna/ https://sandwalk.blogspot.com/2007/07/dna-is-polynucleotide.html
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Molecule DNA - synthesis
Phosphodiester bonds form between the phosphate group attached to the 5' carbon of one nucleotide and the hydroxyl group of the 3' carbon in the next nucleotide.
Regular alternation of the sugar-phosphate-sugar-phosphate motif...Forms the backbone of the polynucleotide: sugar-phosphate (pentose phosphate) backbone.
Chains have chemical polarity: 1 end contains phosphate (5-end), the other contains hydroxyl group (3-end).
Elongation (synthesis) of polynucleotide chain always runs in the direction 5' - 3'.
? ff)
II
o
i— ester bond
phosphodiester -bond
1— ester bond
18 Department of Experimental Biology
https://courses.lumenlearning.com/microbiology/chapter/structure-and-function-of-dna/
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Two types of nucleic acid
DNA
usually double-stranded molecule
Strands are bond by hydrogen bonds between base pairs o adenine - thymine o guanine - cytosine
RNA
usually single-stranded molecule
instead of thymine there is uracil.
DOUBLE-STRANDED SUGAR,*PHOSPHAT£
DEOXVR180SE O
usually single-strandei sugar* phosphate
ought Co.
19 Department of Experimental Biology https://www.thouqhtco.com/dna-versus-rna-608191 M U N I
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Differences between DNA and RNA
Comparison	DNA	RNA
Full Name	Deoxyribonucleic Acid	Ribonucleic Acid
Function	DNA replicates and stores genetic information. It is a blueprint for all genetic information contained within an organism.	RNA converts the genetic information contained within DNA to a format used to build proteins, and then moves it to ribosomal protein factories.
Structure	DNA consists of two strands, arranged in a double helix. These strands are made up of subunits called nucleotides. Each nucleotide contains a phosphate, a 5-carbon sugar molecule and a nitrogenous base.	RNA only has one strand, but like DNA, is made up of nucleotides. RNA strands are shorter than DNA strands. RNA sometimes forms a secondary double helix structure, but only intermittently.
Length	DNA is a much longer polymer than RNA. A chromosome, for example, is a single, long DNA molecule, which would be several centimetres in length when unravelled.	RNA molecules are variable in length, but much shorter than long DNA polymers. A large RNA molecule might only be a few thousand base pairs long.
Sugar	The sugar in DNA is deoxyribose, which contains one less hydroxyl group than RNAs ribose.	RNA contains ribose sugar molecules, without the hydroxyl modifications of deoxyribose.
20 Department of Experimental Biology
https://www.technologynetworks.com/genomics/lists/what-are-the-key-differences-between-dna-and-rna-296719
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Differences between DNA and RNA
Full Name	Deoxyribonucleic Acid	Ribonucleic Acid
Sugar	The sugar in DNA is deoxyribose, which contains one less hydroxyl group than RNA's ribose.	RNA contains ribose sugar molecules, without the hydroxyl modifications of deoxyribose.
Bases	The bases in DNA are Adenine (A'), Thymine (T), Guanine ('G') and Cytosine ('C').	RNA shares Adenine (A), Guanine ('G') and Cytosine ('C') with DNA, but contains Uracil ('U') rather than Thymine.
Base Pairs	Adenine and Thymine pair (A-T) Cytosine and Guanine pair (C-G)	Adenine and Uracil pair (A-U) Cytosine and Guanine pair (C-G)
Location	DNA is found in the nucleus, with a small amount of DNA also present in mitochondria.	RNA forms in the nucleolus, and then moves to specialised regions of the cytoplasm depending on the type of RNA formed.
Reactivity	Due to its deoxyribose sugar, which contains one less oxygen-containing hydroxyl group, DNA is a more stable molecule than RNA, which is useful for a molecule which has the task of keeping genetic information safe.	RNA, containing a ribose sugar, is more reactive than DNA and is not stable in alkaline conditions. RNA's larger helical grooves mean it is more easily subject to attack by enzymes.
Ultraviolet (UV) Sensitivity	DNA is vulnerable to damage by ultraviolet light.	RNA is more resistant to damage from UV light than DNA.
21   Department of Experimental Biology
https://www.technologynetworks.com/genomics/lists/what-are-the-key-differences-between-dna-and-rna-296719
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Base pairing
DNA base pairing
Base pairing is due to hydrogen bonds of opposite bases.
between two strings = duplex
between three strings = triplex
between four strings = quadruplex.
Hydrogen bond acceptors Hydrogen bond donors
O - H-N
H
N.
H-N
O
Thymine or Uracil
1 donor 1 acceptor
N
N
Adenine
1 donor 1 acceptor
N
-i
H-N^PN
N
\
O
H-N
Cytosine
1 donor
2 acceptor
\
H
Guanine
2 donor 1 acceptor
Jack Westin
22  Department of Experimental Biology
https://jackwestin.com/resources/mcat-content/nucleic-acid-structure-and-function/base-pairing-specificity-a-with-t-g-with-c
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Base pairing- tautomerization
(.i) Standard base pairing arrangements
>N—H-\ / N—C
*  V J
O H
Thymine (keto) Adenine (amino)
♦HP*
0---u—n
\
Cylosirie (amino) {b) Anomalous base-pairing arrangements
Guanine (ketoj
CH,
W
H—C
hi —c
4
\
23 Department of Experimental Biology
Standard base-pairing arrangements of the canonical nucleotide isomers.
• Anomalous base-pairing arrangements of the tautomers.
• Tautomerization is the process when a nonstationary proton within the nucleoside quantum tunnels from a common location to a less-common position within the aromatic ring.
• When tautomerization occurs during replication, the DNA sequence will be "misread", and anomalous base-pairing will occur: such as C* with A, or T*.
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https://wwwTesearchgate.net/publication/334974633_Unified_Physics_and_the_Ent^ lement_Nexus_of_Awareness ^
H H
H N--H—t/        M H
4 \
O H
Nucleic Acid is a polymer - macromolecules
Watson - Crick pairing rules
DNA
Adenine Thymine Guanine Cytosine
(amino form)     (keto form)     (keto form)    (amino form)
25 Department of Experimental Biology
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Watson - Crick pairing
H
N—H------O
RNA
Adenine Uracil (amino form)    (keto form)
26 Department of Experimental Biology
Studies in History and Philosophy of Biological and Biomedical Sciences 42 (2011) 119-128
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Chargaff's rules qq g
Rule 1: (SO " ©
•The amount of Adenine- equals the amount of Thymine Purim's =
• The amount of Guanine ~ equals the amount of Cytosine
• The amount of purine = the amount of pyrimidine
A + G _ T + A
Rule2: TVc = 1      "cTg ^ 1
• The amount of A+T / amount of G+C
• This ratio varies among different organisms, but same in different tissues of the same organism.
agggggggaagg   purines     3 + 9    12 9 + 9 18
tcccccc'cttcc   pyrimidines 3 + 9    12 3 + 3     6 is different
(%GC)
27 Department of Experimental Biology https://www.biologyexams4u.com/2021/05/chargaffs-experiment-and-why-is.html MUNI
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Abundance of nucleotides in different organisms
• Erwin Chargaff's Data (1950 - 1951)
		Bat* composition, mole percent				Base ratios	Asymmetry ratio	
								A+T
	A	C	C	T	A/T	C/C	Pu/Py	C + C
Animals								
Man	Kl'l	19 0	19 8	29.4	1.05	1.00	1 04	1.32
BnMB	29.3	.'I 4	21.0	26.3	1.03	1.02	1.03	1.36
Hen	28.8	20.3	21.S	29.2	1.02	0.95	0.97	1.36
Turtle	29.7	220	21.3	27.9	1.05	1.03	1.00	1 31
Salmon	29.7	206	20 4	29.1	1 02	1 02	1 02	1.43
Si-.i urchtn	32.B	17 7	17.3	32.1	1.02	1.02	1.02	1 56
Uocun	29.3	20 5	20.7	20.3	1.00	1 00	1.00	1.41
Planu								
Wheat (term	27.3	22.7	22.6	27.1	1.01	1 00	1.00	1.10
Yeast	31.3	18 7	17.1	32.0	005	1 Iľl	1 00	1.79
Aspergillus niger (mold)	25.0	25.1	2S.0	24.9	1 00	1 00	1 00	1.00
Bacteria								
Ecoli	24.7	26.0	25.7	23.6	1 04	1.01	1.03	0.93
Staphylococcus aureus	308	21 0	19 0	29.2	LOS	1.11	1.07	1.50
Clostridium perfringens	38.9	140	12.6	36.3	1.01	1.09	104	2.70
Brucella abortus	21.0	29.0	28.9	21.1	1 00	1 00	1.00	0.72
Sarcina Jufea	13.4	37.1	37.1	12.4	i.oe	t 00	1 04	0.35
Bacteriophage*								
T7	260	24.0	24 0	26.0	1.00	100	1 00	1 08
	21.3	26.6	27.2	22.9	0.92	1.05	1.00	o -<i
4X174. viral	24.6	24.1	18.5	32 7	0.75	1.30	095	1.34
4VX174. replicative	26.3	22.3	22.3	26 4	1 00	1 00	1.00	1 18
28 Department of Experimental Biology
https://www.eucarpia.eu/tschermak-seysenegg
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Abundance of nucleotides in different organisms
• Plasmodium falciparum (GC% = -20%)
• Streptomyces coelicolor (GC% = 72%)
Organism	%A	%G %C	%T	A/T G/C			%GC		%AT
9X174	24.0	23.3 21.5	31 2	0	77 1	08	44	8	55.2
Maize	268	22.8 23.2	27.2	0	99 0	98	46	1	54.0
Octopus	33.2	17.6 17 6	31.6	1	05 1	00	35	2	64.8
Chicken	28.0	22.0 21.6	28.4	0	99 1	.02	43	7	56.4
Rat	286	21.4 20 5	284	1	01 1	00	42	9	57.0
Human	293	20.7 20.0	30.0	0	98 1	.04	40	7	59.3
Grasshopper 29.3		20.5 20.7	293	1	00 0	99	41	2	58.6
Sea Urchin	32.8	17.7 17.3	32.1	1	02 1	02	35	0	64.9
Wheat	27.3	22.7 22 8	27.1		01 1	.00	45	5	54.4
Yeast	31.3	18.7 17.1	32.9	0	951	.09	35	S	64.4
E. coll	24.7	26 0 25.7	23.6	1	051	.01	51	7	48 3
29 Department of Experimental Biology https://biology.stackexchange.com/questions/43549/is-chargaffs-rule-really-applicable
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Structure of DNA
B-form dsDNA
Complementarity of both strands.
Schematic diagram DNA
Axis of helix
Distance of the backbone from the axis = 1 nM.
Distance of bases is 0,34 nM.
Antiparallelism = direction of phosphodiester bonds 5'-3' and 3'-5'.
Planar character of bases.
Smaller and larger groove = places of protein binding to DNA.
One turn 3.4 nm
(10 5 bp)
Major groove
Minor groove
Zh-0.34
2 nm
30 Department of Experimental Biology
https://www.seekpng.com/ima/u2w7q8u2o0w7e6y3/
Structure of DNA
2 nm
(A)
0.34 nm
\^        hydrogen bond 5'end \ IB)
phosphodiester bond
3'end
• The bases are oriented inside the double helix to the energetically most advantageous arrangement.
• One helix turn accounts for 10.5 base pairs.
• The diameter 2 nm.
• Winding creates a large and small groove in the helix.
• Both twin helix strands are antiparallel and fully complementary.
31   Department of Experimental Biology
https://slideplayer.com/slide/4955288/
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Minor and Major groove of DNA
• The major and minor grooves are opposite each other.
• Each runs continuously along the entire length of the DNA molecule.
• They arise from the antiparallel arrangement of the two backbone strands.
• The grooves are important in the attachment of DNA Binding Proteins involved in replication and transcription.
Minor Major
32  Department of Experimental Biology
https://www.mun.ca/biology/scarr/MGA2_02-07.html
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DNA recognition code
Majorgroove
Major groove
Minor groove
Major groove
Minor groove
Major groove
Minor groove
Minor groove
• H-bond acceptor
• H-bond donor
• methyl group
• other
A T
G C
T A
C G
A T
G C
T A C G
Majorgroove     Minor groove
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33  Department of Experimental Biology Int J Mol Sci. 2014 Jul; 15(7): 12335-12363. or»T
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Strands in DNA helix are in antiparallel orientation
Adenine Thymine
• Two strands of DNA have the same helical geometry however base pairing holds the strands together with opposite polarity.
• The 5' end of a strand is paired with the base of the end of the other strand 3'.
• This is called anti-parallel orientation, the consequence that adenine and thymine pair with each other and guanine and cytosine pair with each other.
34 Department of Experimental Biology
http://dnastructureandreplication.weebly.com/enzymesanti-parallel.html
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Winding of DNA
• Rosypal (1998): "If we place the thumb of right hand in the direction of the Double helix axis, and other fingers point direction of its ascent/climbing - it is a right-handed double helix. Left-handed double helix corresponds to a similar rule of the left hand.,,
• There is an experiment proving the principle underlying the Vester-Ulbricht hypothesis that the primarily left-handed spinning electrons in cosmic rays could have preferentially destroyed left-handed precursors of DNA, leaving only right-handed DNA. The sculpture illustrates DNA's right-handed double helix.
tax 4-1
I»frflrflgcf Prinf'pStf pf 8T«*f mürry, fifth Edition
■'■ iOOfl W.H, Freeman and Company
35 Department of Experimental Biology https://twitter.com/van_oijen/status/1099122913234477056?lang=de
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Conformation of dsDNA or dsRNA
• Conformation = spatial arrangement of the biomacromolecule into a structure, which is the most energy-efficient under the given conditions.
• DNA conformation depends on: o nucleotide sequence
o water content in the environment o ion force of the environment.
36 Department of Experimental Biology
https://www.mun. ca/biology/scarr/Beadle_&_Tatum_Experi ment.html
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Forms of DNA
FORMS
• B-DNA: right-handed, in aqueous solution and at normal salt concentrations.
• A-DNA: right-handed, with 11 bp per turn, in dehydrated samples.
• Z-DNA: left-handed, with 12 pb per turn, occurrence in double helixes GC-rich, function unclear in living systems.
A-form RNA B-form DNA Z-form DNA
37 Department of Experimental Biology
http://www.tulane.edu/~biochem/nolan/lectures/rna/DNAstruc2001.htm
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Forms of DNA
	8 form DNA	A form DNA	Z form DNA
Helical sense	Right handed	Right handed	Left handed
Major groove	Present	Present	Absent
bp/helical tern	10.5	11	12
Glycosyl bond conformation	Anti	Anti	Anti (for pyrirnidines) and syn (for Purines)
Helix rise/bp	3.4A	2.6A	3.7A
Base tilt	6°	20°	7°
			
Structure	¥		
		I	1
38 Department of Experimental Biology
https://geneticeducationxojn/dna-deoxyribonucleic-acid-definition-structure-function-evidence-and-types/
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A-form of DNA
A-form
• Most RNA and RNA-DNA duplex in this form.
• Shorter, wider helix than B.
• Deep, narrow major groove not easily accessible to proteins.
• Wide, shallow minor groove accessible to proteins, but lower information content than major groove.
• Favored conformation at low water concentrations.
• Base pairs tilted to helix axis.
A form
39 Department of Experimental Biology
https://microbenotes.com/different-forms-of-dna-b-form-a-form-z-form/
B-form of DNA
B-form
• Most common DNA conformation in vivo.
• Narrower, more elongated helix than A.
• Wide major groove easily accessible to proteins.
• Narrow minor groove.
• Favored conformation at high water concentrations (hydration of minor groove seems to favor B-form).
• Base pairs nearly perpendicular to helix axis.
B form
Department of Experimental Biology
https://microbenotes.com/different-forms-of-dna-b-form-a-form-z-form/
Z- form of DNA
Z-form
• Helix has left-handed sense.
• Can be formed in vivo, given proper sequence and superhelical tension, but function remains obscure.
• Narrower, more elongated helix than A or B.
• Major "groove" not really groove.
• Narrow minor groove.
Zform
Department of Experimental Biology
https://microbenotes.com/different-forms-of-dna-b-form-a-form-z-form/
Z- form of DNA
Z-form
• Conformation favored by high salt concentrations, base substitutions, but requires alternating purine-pyrimidine sequence.
• Base pairs nearly perpendicular to helix axis.
• GpC repeat, not single base-pair.
• Zigzag backbone due to C sugar conformation compensating for G glycosidic bond conformation.
Zform
42  Department of Experimental Biology
https://microbenotes.com/different-forms-of-dna-b-form-a-form-z-form/
Forms of DNA
43 Department of Experimental Biology
https://microbenotes.com/different-forms-of-dna-b-form-a-form-z-form/
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DNA Organization
44 Department of Experimental Biology
https://www.pngwing.com/en/free-png-ptbih
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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5'
"O O
OH H
45 Department of Experimental Biology
Primary structure of DNA
• Primary structure is sequence of bases in the nucleic acid chain gives the primary structure of DNA or RNA.
• The sequence of bases is read in a 5' —► 3' direction, so that you would read the structure in the next figure as ACGT
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
AC GB pi Of
Secondary structure of DNA
The base-pairing of complementary nucleotides gives the secondary structure of a nucleic acid. In a double-stranded DNA or RNA, this refers to the Watson-Crick pairing of complementary strands. In a single-stranded RNA or DNA, the intramolecular base pairs between complementary base pairs determines the secondary structure of the molecule.
Sacwdaiy ■ Basů pa>ra
46 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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Tertiary structure of DNA
Acceptor Sierr
□ toop
variable
Anlüüdiüi stsrn
Anticodon loop
The tertiary structure of a nucleic acid refers to the three-dimensional arrangement of the nucleic acid - that is, the arrangement of the molecule in space, as in the tertiary structure of tRNA.
k'l-.lf.    r--:c.) i:
47 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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Quaternary structure of DNA
Quaternary structure refers to the large shapes and structures that can be made by nucleic acids.
DNA - histone = nucleosome
RNA - protein = ribosome
Yeast
H3J
48 Department of Experimental Biology
https://www.pngwing.com/en/free-png-ptbih Cell, Vol. 107, 373-386, November 2, 2001
Organization of dsDNA
• The DNA double helix may be arranged in space, in a tertiary arrangement of the strands.
• The two strands of DNA wind around each other. In a covalently closed circular DNA (cccDNA), this means that the two strands can't be separated.
49 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
NucleicAcids Research, 2014, Vol. 42, No. 20, 12404-12414
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Organization of dsDNA
Open circular Super colied (OC) dsDNA   (SC) dsDNA
oc
sc
50 Department of Experimental Biology
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Organization of dsDNA
In closed circular DNA the strands can't be separated, the total number of turns in a given molecule of cccDNA is a constant, called the Linking Number, or Lk.
The linking number of a DNA is an integer and has two components,
Twist (Tw), or number of helical turns of the DNA.
Writhe (Wr), number of times the double helix crosses over on itself (these are the supercoils).
• Because Lk is a constant, the relationship can be shown by the equation:  Lk = Tw + Wr
1 5 10 15 20 25
xxxxxx?bcx>ooobocoocbc<xxxxxxxxxx
20,
Close ends to make a c*cle
(a)
Up- 25. 7W* 25. Wr* 0 Relaxed orcle
51   Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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Twist = -1, Writhe = 0.
Twist = 0, Writhe = -1.
Q^?720°
Twist = -2, Writhe = 0.
Twist = 0, Writhe = -2
I
USD 360°
Twist = +1, Writhe = 0.
Twist = 0, Writhe = +1.
Organization of dsDNA
The simple figure eight is the simplest supercoil (writhe), and is the shape a circular DNA assumes to accommodate one too many or one too few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over or underwound.
Twist = +2, Writhe = 0.
Twist = 0, Writhe = +2.
CP
Twist = 0, Writhe = -4.
Plectonemic
Toroidal
52  Department of Experimental Biology
• Twist and writhe are interconvertible.
• Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling.
https://bioJibretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless) MUNI /7%3A_Microbial_Genetics/7.02%3A_Prokaryotic_Genomes/7.2B%3A_Supercoiling SCI
Organization of dsDNA
Negative supemehx Lks 23. Tw- 23. Wi~ 0 (Right-handed) Unwound circle
• Normally, this DNA would have a linking number equal to 25, so it is underwound.
• The DNA double helical structures have the same value of Lk; however, the DNA can be supercoiled, with the two "underwindings" taken up by the negative supercoils.
• This is equivalent to two "turns'-worth" of single-stranded DNA and no supercoils.
53 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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Organization of dsDNA
• As a general rule, the DNA of most organisms is negatively supercoiled.
Positive superhelix
Negative superhelix
54 Department of Experimental Biology
https://slideplayer.com/slide/4137430/ https://quizlet.com/203493874/lecture-33-flash-cards/
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Organization of dsDNA
• Increasing number of superhelixes of plasmid DNA.
55 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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DNA - topoisomerases
• DNA - topoisomerases - alter Lk, the linking number of a DNA, by a bond breaking and rejoining process.
• Catalyze the formation of transitional breaks in DNA
• Break ("nick") = breaking of the phosphodiester bond between     . Cut in one DNA strand is catalyzed by neighboring bases. topoisomerase I.
• Cuts in both DNA strands are catalyzed by topoisomerase II = DNA-gyrase.
56 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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DNA - topoisomerase I
• Type I topoisomerases (sometimes called "nicking-closing enzymes") carry out the conversion of negatively supercoiled DNA to relaxed DNA in increments of one turn.
• Type I , increases Lk by increments of one to a final value of zero.
• Type I topoisomerases are energy independent, because they don't require ATP for their reactions.
• Anti-tumor drugs, including Camptothecin, target the eukaryotic topoisomerase I.
57 Department of Experimental Biology
t
***c *ou*oubo*» DNA M mtniiit otoůt vtftodtn ktinjitm bono
jiiixie:
^ OHMopootwia i
M kovilifltni pApopft k loafctu * DM. imj p/enA
I ONA
Kon» CMwífOuDWCt DNA M oyni fnonou vzá|Vniné Otteft
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tiilodiima) it iiiDi
ftmtoutXMict OHA. WiDNA-kpoizonwtal v púvooni tome
HO
: nun::
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DNA - topoisomerase I
• Releases superhelix tension from the superhelix DNA.
• Topoisomerase covalently attaches to one of the phosphates in DNA, o cuts DNA strand can rotate around its longitudinal axis,
o the strand tension/pressure is relieved,
o double helix restoration and enzyme is released.
Double stranded helix v
Double helix
(-1
Binding of Topo I
Topo I
\
Nick DNA; form covalent DNA-phosphotyrosine bond
under other strand and reseal DNA
Pass cut 3' end
Dissociation of TopoI
3 negative supercoils
2 negative supercoils
58 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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DNA - topoisomerase II (DNA Gyrase)
• Type II topoisomerases (sometimes called DNA gyrases) reduce Lk by increments of two.
• These enzymes are ATP-dependent and will alter the linking number of any closed circular DNA.
• The antibiotic Nalidixic acid, which is used to treat urinary tract infections, targets the prokaryotic enzyme.
• Type II topoisomerases act on naturally occurring DNAs to make them supercoiled.
59 Department of Experimental Biology
https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures
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Differences between topoisomerases I and II
Supercoiled DNA
Type I
Type II ->
60 Department of Experimental Biology
Nick in one strand
Nick in both strands
• the one strand moves above the other one in the cut and merges:
• L is reduced by 1
both strands move above the break and merges: + ATP for function
• L is reduced by 2
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https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/genomics/sequencing/sanger- C P sequencing
DNA organization in regulation of trascription
Eukaryotic DNA is linear and double-stranded.
• It binds to protein scaffolding.
• Generates superhelixes, solenoid loops and relaxed areas.
negative supercoiling
left-handed mitleosomc
0«
podive tupefcoillrpg
right-handed nixleoiome
topological^ associating domain (TAD)
transcription factories (condensates) / "I
supercoiled DNA
promoter enhancer cohesin complex CTCF
DNA topoisomerase II
61   Department of Experimental Biology
Nanoscale. 2017 Feb 2; 9(5): 1862-1870.
Nucleic Acids Research April 2020 48(9):4614-4626
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DNA sequences adopting alternative structures
• Unique DNA sequence: ....AATGCTGATGTCTGACTCGGA...
• Repetitive sequences or repeat.
• Terms: unit of repetition, length of unit of repetition, frequency of repetition.
• Example: ATG... ATG....ATG....ATG...unit = ATG, length = 3 nucleotides, frequency = 4x.
• Tandem repeats - tied tightly to each other "head to toe".. ATGCATGCATGC
• Direct repetition (5....ATGC.....ATGC.....3) repeats on the same strings in
the same direction (5' 3).
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62  Department of Experimental Biology Dig Dis Sci. 2015 Aug; 60(8): 2230-2231. p> o t
DNA sequences adopting alternative structures
Inverted repetition: repeated on the second string in the reverse direction -potential for creating a hairpin or hairpin with a loop.
5'...ATGCGCAT...3' palindrome (hairpin/hairpin with loop)
3'...TACGCGTA...5'
5 ... ATGCXXXXXGCAT... 3' 3'... TACGYYYYYCGTA... 5'
hairpin with loops (within dsDNAthe cross structure is established)
LTR - long terminal repeat
5'-ATGC...GCAT............
3'-TACG...CGTA............
Palindrome
T T A G C A C G T G C T A A "I   I   I   I   I   I T
J_I   I   I_l_l_L      I   I   I   I   I I
ATGC. TACG.
GCAT-3' CGTA-5'
AATCGTGCACGATT
Mirror repeat
TTAGCACCACGATT
_l_l   i   l   1   l   l l_......
AATCGTGGTGCTAA
63 Department of Experimental Biology
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Sheme of hairpin structure
eI ._TGCGAT ACTC ATCGC A_.
5 4      TTT I I I I TTI      * 3
5'«
TTT
TTT
Hairpin
* 3'
5'«-
3'«-
_TGCGATACTCATCGCA
TTT I I I I
''''
nr
J_LL
ACGCTATGAGTAGCGT
3*4
Cruciform
-► 3'
5'
64 Department of Experimental Biology
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Denaturation and Renaturation of DNA and RNA
• Denaturation of dsDNA = transformation of dsDNA into ssDNA.
• Renaturation of ssDNA = transformation of ssDNA into dsDNA.
• Induction of denaturation:
o by increasing the temperature of the solution,
o by changing the pH from neutral to alkaline or acidic.
• Occurs in vitro and naturally also in vivo.
Native double Strands Separated Double helix
helix unwinding strands reformed
■   FIGURE 9.1 9 Helix tin winding in DNA denaturation. The double helix unwinds when DNA is denarured, wiih eveiuual separation oFuhe strands. The double helix is re-formed on rena-tumtion ■willi slow cooling and annealing.
65  Department of Experimental Biology
https://www.brainkart.com/article/Denaturation-of-DNA_27539/
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Denaturation and Renaturation of DNA and RNA
• dsDNA denaturation is manifested by hyperchromic effect, which means increased absorbance of UV-light with a wavelength 260 nm.
• Value Tm or melting point = temperature, in which 50% of dsDNA molecules are denatured.
• Tm depends on the content of the bases.
Temperature. "C
66 Department of Experimental Biology
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Denaturation curve
Other options for determining %GC:
• Ultracentrifugation in CsCI
• HPLC.
100
Tm= 69,3 * 0,41 (CC).
GC =
_ Tm - 69,3
0,41
67 Department of Experimental Biology
50
Poly (AT)
Naturally occurring DNA with A/Tand G/C base pairs
—r-1-1 i
10        30 50
PolylGQ
Temperature (CC)
• GC = molar fraction of guanine and cytosine in DNA,
• 69.3 and 0.41 are empirically the coefficients laid down,
• pro poly(AT) Tm = 69,3.
https://sandwalk.blogspot.com/2007/12/dna-denaturation-and-renaturation-and.html
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Hybridization of DNA and RNA
• Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing.
• The more hybridizing molecules coincide in sequences, or the higher their sequential homology, the greater it is the probability of their hybridization.
Double Stranded Single Stranded Double Stranded
68  Department of Experimental Biology DNA-Based Storage: Trends and Methods, July 2015, DOI: 10.1109/TMBMC.2016.2537305
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Hybridization of DNA and RNA
Use for evaluation of the degree of sequential/structural similarity of DNA without sequencing.
Prepare DNA
Separate strands and        Cool and allow to re-anneal mix
Sample 1
Sample 2
MXMM>
Sample 3
69 Department of Experimental Biology
High melting temperature = Samples 1 and 2 are closely related
Low melting temperature = Samples 1 and 3 are not closely related
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Fly
T
-G T A G
A
C A T
C
- G C-l
■C G
A C A
C
T
GH
T G C
-he    G H C GJ
Hybridization of DNA and RNA
Human
■-T A -i
G G
A T
A T
G C -
H A -
C G
- A T -
-C G -
A T
T A
T A
KG CH
Chimp
T
G A A. G A C A C A T G
A -i
C T T
C-T
Q-T
T 4. QH
T
A
cH
G C -C    G ■■
Double-helical DNA from two species is unwound (denatured), cut, and mixed.
Complementary pieces bind (called reannealing), forming some hybrid DNAs—one strand from each species.
Then the temperature is raised and the rate of interspecies double-helix separation determined.
The higher the temperature needed to separate the hybrid DNA, the more similar the DNA sequences must be.
Fly/human HurňarVctnimp (leas conservation) (more conservation)
70 Department of Experimental Biology httpsV/www.sciencedirectxom/topics/medicine-and-dentisti^/dna-hybridization
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Usage of Hybridization in the reasearch
Identification of specific DNA a RNA sequences.
Estimation of their structural similarity. PCR.
Transcription in vitro. FISH.
DNA hybridization
single-stranded DNA probes for gene A
hybridization in 50% formamide at 42°C
only A forms stable double helix
ieA /
mixture of single-stranded DNA molecules
JL
hybridization in 50% formamide at 35°C
imperfect J base-pairing
A
A, C, and E all form stable double helices
71   Department of Experimental Biology
Figure 8-25. Molecular Biology of the Cell, 4th Edition.
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THANK YOU FOR YOUR ATTENTION
IF I AM LOST IN TRANSLATION
72  Department of Experimental Biology
base
{here: guanine}
q
n _>NH
:ibose
O OH phosphate 0=P—0-, Ô"
JUST BLAME MY RNA
https://www.jokejive.com/topic/molecular+biology#&gid=1&pid=8
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