Оберемок В.В. Справочник по ДНК-модифицирующим энзимам и связанным с ними методам молекулярной генетики (на русском и английском языках)
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1,5 200-3000
3,0 25-1000
5,0 10-300
Таблица II. Общие физические константы
Название Константа
Число Авагадро 6.022x10
23
моль
-1
Константа Планка
6.626x10
-34
Дж·с
Константа Больцмана
1.38x10
-23
Дж·K
-1
Постоянная идеального газа
8.314 Дж/моль·K
Скорость света 2.9979x10
8
м/с
Единица атомной массы 1.66x10
-27
кг
*Пересчёт концентрации олигонуклеотидов
Молекулярная масса:
MW =333xN
Концентрация олигонуклеотидов:
C (µM/ или пмоль/мкл) = A
260
/0.01xN;
C (нг/мл) = A
260
xMW/0.01xN;
MW – молекулярная масса, A
260
– поглощение при 260 нм, N – число
оснований
*ДНК/белок пересчёт
1 кб ДНК= 333 аминокислоты = 37 кДа
*Температура плавления двухцепочечной ДНК и
олигонуклеотидов
Для олигонуклеотидов короче, чем 25 п.о., “Правило Уоллеса ”
T
m
(в
o
C) = 2(A+Т) + 4(Г+Ц), где
(A+Т) – сумма адениновых и тиминовых оснований в
олигонуклеотиде, а (Г+Ц) – сумма гуаниновых и цитозиновых
оснований в олигонуклеотиде.
Для двухцепочечной ДНК < 100 п.о. длиной
T
m
(в
o
C) = 81.5
o
C+16.6 (log
10
[Na
+
])+0.41(%[Г+Ц]) –675/n –1.0m, где
n – число оснований в олигонуклеотиде;
m – процент несоединенных водородной связью пар оснований
*Часто используемые буферные растворы
1 M Трис-HCl [трис(гидроксиметил) аминометан]:
Трис 121.1 г
H
2
O до 800 мл
Доведите до желаемого pH концетрированной HCl.
Смешайте и доведите H
2
O до литра
31
1X TБЕ (Трис-боратный-ЭДТА) буфер для электрофореза:
Трис 108 г
Борная кислота 55 г
0.5 M ЭДТА (pH 8.0) 40 мл
H
2
O до 1 л
1X TE (Tрис-ЭДТА) буфер, pH 7.4, 7.6 или 8.0, на литр:
1 M Трис, pH 7.4, 7.8, 8.0 108 г
0.5 M ЭДТА (pH 8.0) 2 мл
H
2
O до 1 литра
*Выражение концентраций реагентов
Молярность
M (моль/л) = число молей растворённого вещества/число литров
раствора
Концентрация
Концентрация, С (%) = вес растворённого вещества (г)x100%/общий
вес раствора (г)
Ионная сила
Ионная сила, I, раствора – это функция от
концентрации всех ионов, присутствующих в
растворе, где c
i
– молярная концентрация иона
i (моль·дм
-3
), z
i
– заряд данного иона;
суммируются все ионы в растворе.
Таблица III. Размеры геномов
Вирусы Длина в
основаниях
Прибл. атомная
масса, Дa
Бактериофаг φX174 5,380 3.5x10
6
ВИЧ 9,181 3.1x10
6
Коронавирус SARS (атипичная 29,751 1x10
7
32
пневмония)
Бактерии
Agrobacterium tumefaciens C58-
Cereon
4.91x10
6
3.19x10
9
Staphylococcus aureus MW2 4.86x10
6
3.02x10
9
Streptococcus pneumoniae R6 2.04x10
6
1.33x10
9
Эукариоты
Anopheles gambiae
PEST (малярийный москит)
2.78x10
8
1.80x10
11
Caenorhabditis elegans
(круглый червь)
1.21x10
7
7.86x10
9
Drosophila melanogaster 1.37x10
8
8.9x10
10
Homo sapiens 3.15x10
9
2.07x10
12
Saccharomyces cerevisiae
S288C (почкующиеся дрожжи)
1.21x10
7
7.86x10
9
Plasmodium falciparum 3D7
(малярийный плазмодий)
2.29x10
7
1.49x10
10
Таблица IV. Атомные числа и молекулярные массы некоторых
химических элементов
H C N O Na Mg P S Cl Ca
Атомное число 1 6 7 8 11 12 15 16 17 20
Атомная масса 1 12 14 16 23 24.3 31 32 35.4 40
Атомная масса– г/моль
DNA POLYMERASES
DNA polymerase I
A template-dependent DNA polymerase, catalyzes 5`→3`
synthesis of DNA. The enzyme also exhibits 3`→5` exonuclease
33
(proofreading) activity, 5`→3` exonuclease activity and ribonuclease
H activity.
Features:
1) incorporates modified nucleotides (e.g., biotin-, digoxigenin-,
fluorescently-labeled nucleotides);
2) active in buffers for restriction enzymes, PCR.
Applications:
1) DNA labeling by nick-translation in conjunction with DNase;
2) second-strand synthesis of cDNA in conjunction with RNase H.
10X Reaction buffer
500 mM Tris-HCl (pH 7.5 at 25
o
С), 100 mM MgCl
2
, 10 mM DTT.
Inhibition and inactivation
Inhibitors: metal chelators; inactivated by heating at 75
o
С for 10 min or by
addition of EDTA.
Fig. 1. Scheme of DNA polymerase I activity: I – synthesis; II –
3`→5` exonuclease activity; III – 5`→3` exonuclease activity; a – DNA-
polymerase I; b – deoxyribonucleoside monophosphates
34
Fig. 2. Structure of DNA Polymerase I
* Interesting details. Fluorescein is a synthetic organic compound
available as a dark orange/red powder soluble in water and alcohol. It is
widely used as a fluorescent tracer for many applications. Fluorescein-
labelled nucleotides can be imaged using FISH (fluorescence in situ
hybridisation), or targeted by antibodies using immunohistochemistry.
*Lab Shelf
EDTA –
ethylenediaminetetraacetic acid. It is widely used
to dissolve scale. Its usefulness arises because of
its role as a hexadentate ("six-toothed") ligand
and chelating agent, i.e. its ability to "sequester"
metal ions such as Mg
2+
,Ca
2+
and Fe
3+
. After
being bound by EDTA, metal ions remain in
solution but exhibit diminished reactivity.
Tris – tris(hydroxymethyl)aminomethane. Tris
is extensively used in biochemistry and
molecular biology. The useful buffer range for
tris (7-9) coincides with the typical
physiological pH of most living organisms.
This, and its low cost, make tris one of the
most common buffers used in the
biology/biochemistry lab.
DTT – dithiothreitol. A common use of DTT is
as a reducing
agent for thiolated
DNA. The
terminal sulfur
atoms of thiolated
DNA have a tendency to form dimers in
35
solution, especially in the presence of oxygen.
Dimerization greatly lowers the efficiency of
subsequent coupling reactions such as DNA
immobilization on gold in biosensors.
Klenow fragment
Klenow fragment is the large fragment of DNA polymerase I.
It exhibits 5`→3` polymerase activity and 3`→5` exonuclease
(proofreading) activity, but lacks 5`→3` exonuclease activity of
DNA Polymerase I.
Features:
1) incorporates modified nucleotides (e.g., biotin-, digoxigenin-,
fluorescently-labeled nucleotides);
2) active in buffers for restriction enzymes, PCR, T4 DNA Ligase.
Applications:
1) DNA blunting by fill-in 5`-overhangs;
2) random primed DNA labeling;
3) labeling by fill-in 5`-overhangs of dsDNA;
4) DNA sequencing by the Sanger method;
5) site-specific mutagenesis of DNA with synthetic nucleotides;
6) second strand synthesis of cDNA.
10X Reaction buffer
500 mM Tris-HCl (pH 8.0 at 25
o
С), 50 mM MgCl
2
, 10 mM DTT.
Inhibition and inactivation
Inhibitors: metal chelators; inactivated by heating at 75
o
С for 10 min or by
addition of EDTA.
Klenow fragment, as well as DNA polymerase I, requires:
1) template;
2) primer;
3) free 3`-OH end;
4) deoxynucleoside triphosphates.
*Interesting details. Klenow fragment, as well as DNA polymerase
I, catalyzes the incorporation of dNTPs at a maximal rate of 20 nt/sec
(approximately).
T7 DNA polymerase
A template dependent DNA polymerase, catalyzes DNA
synthesis in the 5`→3` direction. It is highly processive DNA
36
polymerase allowing continuous synthesis of long stretches of DNA.
The enzyme also exhibits a high 3`→5` exonuclease activity towards
single- and double-stranded DNA.
Features:
1) strong 3`→5` exonuclease activity;
2) active in buffers for restriction enzymes.
Applications:
1) purification of covalently closed circular DNA by removal of
residual genomic DNA;
2) primer extension reactions on long templates;
3) DNA 3`-end labelling;
4) strand extensions in site-directed mutagenesis;
5) fill-in blunting of 5`-overhang DNA;
6) second strand synthesis of cDNA;
7) in situ detection of DNA fragmentation associated with apoptosis.
10X Reaction buffer
400 mM Tris-HCl (pH 7.5 at 25
o
С), 100 mM MgCl
2
, 10 mM DTT.
Inhibition and inactivation
Inhibitors: metal chelators, modification reagents (acetic anhydride, N-
ethylmaleimide inactivate the 3`→5` exonuclease activity but not the
polymerase activity); inactivated by heating at 75
o
С for 10 min.
Pfu DNA polymerase
A thermostable enzyme of approximately 90kDa isolated
from Pyrococcus furiosus. The enzyme replicates DNA at 75
o
C,
catalyzing the polymerization of nucleotides into duplex DNA in the
5´→3´ direction in the presence of magnesium. Pfu DNA
Polymerase also possesses 3´→5´ exonuclease (proofreading)
activity.
Features:
1) base misinsertions that may occur during polymerization are
rapidly exercised by the proofreading activity of the polymerase;
2) active in buffers for restriction enzymes.
3) Pfu DNA polymerase-generated PCR fragments are blunt-ended.
Applications:
1) sequencing;
2) primer extension reactions.
37
Reaction buffer
10 X Buffer: 200 mM Tris-HCl (pH 8.8); 20 mM MgSO
4
, 100 mM KCl; 100
mM (NH
4
)
2
SO
4
; 1% Triton X-100; 1 mg/ml nuclease-free BSA.
Inhibition and inactivation
Inhibitors: EDTA, chelating agents.
*Lab Shelf
Triton X -100 – polyethylene glycol
p-
(1,1,3,3-
tetramethylbutyl)-phenyl ether is a
nonionic surfactant. Can be used in DNA
extraction as part of the lysis buffer. This
compound works by disrupting non-
covalent bonds in the proteins.
* Interesting details. Pfu DNA Polymerase exhibits the lowest error
rate of any thermostable polymerase; reaction rate - is 2kbp/min.
Taq DNA polymerase
Catalyzes the incorporation of dNTPs into DNA. It requires a
DNA template, a primer terminus, and the divalent cation Mg
2+
. Taq
DNA polymerase contains a polymerization dependent 5'→3'
exonuclease activity. It does not have a 3'→5' exonuclease and thus
no proofreading function. Despite this, the enzyme synthesizes DNA
in vitro with reasonable fidelity.
Features:
1) strong 5`→3` exonuclease activity;
2) 30% of amplification products have adenosine monophosphate
overhangs;
3) active in buffers for restriction enzymes.
Applications:
1) PCR. Taq is stable up to 95°C (isolated from Thermus aquaticus);
thus it is not necessary to replenish the enzyme in a 35 cycle PCR .
The maximal enzyme activity is between 70-75°C which
minimizes secondary structure of the template and results in high
38
polymerization yield. Annealing temps can be chosen from 30-
70°C;
2) Cycle sequencing. It gives more uniform bands than Klenow
fragment. The high temperature extension and cycling permits
difficult sequences (hairpins) to be sequenced more readily. It can
read through homopolymer sequences (i.e. dG:dC) better than
Klenow.
Reaction buffer
The enzyme can be diluted in 10 mM Tris (pH 7.6), 0.5 mg/ml BSA, 0.1
mM DTT, 0.1 mM EDTA. Use diluted enzyme the same day.
Inhibition and inactivation
Inhibitors: bromophenol blue, urea, DMSO, formamide and SDS all inhibit
Taq to some extent (xylene сyanol (0.02%) does not inhibit this enzyme).
Taq is inhibited 47% at a concentration of 10% DMSO. It can still be used
for PCR and cycle sequencing of GC rich DNA in the presence of 10%
DMSO if the amount of Taq is doubled.
Reaction mixture for a typical PCR amplification:
1) 5х PCR-buffer – 5 µl;
2) MgSO
4
, 50 mМ – 1,5 µl;
3) H
2
O (distilled) – 3 µl;
4) dNTP-mіx, 2 mM – 2,5 µl;
5) T
aq
-buffer, 5 u/ µl – 0,5 µl;
6) mineral oil – 10,5 µl;
7) 2 primers, 3,3 ОD/mol – по 1 µl;
8) ТЕ-buffer with DNA – 5 µl.
*Lab Shelf
Formamide – it is a clear liquid which is miscible with
water and has an ammonia-like odor. In
its pure form, it dissolves many ionic
compounds that are insoluble in water,
so it is also used as a solvent.
Formamide is also used as an RNA
stabiliser in gel electrophoresis by deionizing RNA. In
capillary electrophoresis, it is used for stabilizing (single)
strands of denatured DNA.
DMSO - dimethyl sulfoxide. This colorless liquid is an
important polar aprotic solvent that dissolves both polar
and nonpolar compounds and is miscible in a wide range
of organic solvents as well as water. It
39
has a distinctive property of penetrating the skin very
readily, so that one may taste it soon after it comes into
contact with the skin. Its taste has been described as
oyster- or garlic-like. DMSO is used in PCR to inhibit
secondary structures in the DNA template or the DNA
primers. It is added to the PCR mix before reacting, where
it interferes with the self-complementarity of the DNA,
minimizing interfering reactions.
SDS – sodium dodecyl sulfate. It can be
used to aid in lysing cells during DNA
extraction and for unraveling proteins.
SDS is commonly used in preparing
proteins for electrophoresis. This
compound works by disrupting non-
covalent bonds in the proteins,
denaturing them, and causing the
molecules to lose their native shape
(conformation). Also, anions of SDS
bind to the main peptide chain at a ratio
of one SDS anion for every two amino
acid residues. This effectively imparts a negative charge
on the protein that is proportional to the mass of that
protein (about 1.4 g SDS/g protein).
BSA – bovine serum albumin. In restriction digests, BSA is used to stabilize
some enzymes during digestion of DNA and to prevent adhesion of the
enzyme to reaction tubes and other vessels. This protein does not affect
other enzymes that do not need it for stabilization. BSA is also commonly
used to determine the quantity of other proteins, by comparing an unknown
quantity of protein to known amounts of BSA.
Polymerase chain reaction
The polymerase chain reaction is a technique for quickly
"cloning" a particular piece of DNA in the test tube during 30-40
cycles. Polymerase chain reaction was invented by US biochemist
Kary Mullis in 1983. Wide level of opportunities, cheapness and
40