III. Chromosome replication is
coordinated with cell division
III. Chromosome replication is
coordinated with cell division
2. Timing of initiation of replication
(1) The bacteria are synchronized, and
then the amount of DNA is
measured.
(2) Fig.1.20 shows the timing of DNA
replication during the cell cycle,
with two different generation time.
III. Chromosome replication is
coordinated with cell division
I, the time from replication initiated to a new round begins.
C, the time of entire chromosome replication. (40’ at 37 oC)
D, the time from replication completed to cell division occurs. (20’ at 37 oC)
(1) C, D remain the same independent of the growth rate.
(2) I gets shorter when cell’ growth is fast, and cell has short generation
time (the time it takes a newborn cell to grow and divides.
(3) The initiation of chromosome replication occurs each time the cell achieves a
certain mass, the initiation mass.
III. Chromosome replication is
coordinated with cell division
4. The timing of initiation of chromosome replication is tied to the
intracellular concentration of DnaA protein.
5. Dam methylase can methylate two A’s in GATC/CTAG
sequence
(1) This sequence repeated 11 times in 245 bp oriC region.
i. Immediately after an oriC region has been used to initiate
replication, the GATC/CTAG sequences in oriC are
hemimethylated.
ii. The hemimethylated oriC region is seqestered by binding to the
membrane (may help by protein SeqA) , a process that renders it
nonfunctional for the initiation of new rounds of replication.
iii. This sequence also exists in DanA promoter, and no DnaA is synthesized
unless these sequences are fully methylated.
(2) There is direct evidence to support the role of methylation in oriC
sequestration and regulation of DnaA protein synthesis after initiation.
III. Chromosome replication is
coordinated with cell division
IV. Some methods for DNA study
Nucleic acids hybridize by base
pairing
· A crucial property of double helix is the ability to separate the two
strands without disrupting covalent bond. This makes it possible for
the strands to separate and reform under physiological conditions at the
( very rapid) rates needed to sustain genetic functions.
· Heating and some chemicals (e.g., NaOH) cause the two strands of a DNA
duplex to separate.
· Complementary single strands can renature when the temperature is
reduced.
· Denaturation and renaturation/hybridization can occur with DNA-DNA,
DNA-RNA, or RNA-RNA combinations, and can be intermolecular or
intramolecular.
· The Tm is the midpoint of the temperature range for denaturation.
· The ability of two single-stranded nucleic acid preparations
to hybridize is a measure of their complementarity.
DNA Melting
• With heating, noncovalent
forces holding DNA strands
together weaken and break
• When the forces break, the
two strands come apart in
denaturation or melting
• Temperature at which DNA
strands are ½ denatured is
the melting temperature or
Tm
•
•
•
The amount of strand separation, or melting, is measured by
the absorbance of DNA solution at 260 nm
GC content of DNA has a significant effect on Tm with higher GC
content meaning higher Tm
noncovalent forces – hydrophobic property of bases, hydrophilic
property of backbone and hydrogen bonds
Denatured single strands of DNA can
renature to give the duplex form.
Filter hybrydization
Calculation of Tm for a DNA
molecule
1.Tm = (G+C)%/2.44 + 81.5 + 16.6log[Na+]
2. For oligonucleotide:
Tm = 4 (G+C) + 3 (A+T)
3. In the presence of foramide:
minus  0.67 0C per %
4. High stringency and low stringency
Measurement of nucleic acid
concentration
1. Hypochromic effect: The heterocyclic rings of
nucleotides absorb light strongly in UV range (with
maximum close to 260 nm that is characteristic for each base).
But the absorption of DNA itself is 40% less than would be
displayed by a mixture of free nucleotides of the same
composition.
2. Concentration of nucleic acid in a solution (ug/ml):
DNA: OD260 X 50 X (dilution factor)
RNA: OD260 X 40 X (dilution factor)
Oligo: OD260 X 30 X (dilution factor)
Restriction enzymes
Restriction and modification
(限制與修飾 )
Restriction enzymes
A. Cleave DNA at specific sequences (recognition
sequence, cutting site ).
(一種可以針對特定DNA序列進行辨認與切割的蛋白質)
B. Firstly discovered by Arber and Smith (late 1960s)
C. Boyer (1969) first isolated EcoRI
D. Different restriction enzymes may share the same
recognition sequence although they do not necessary
cut at precisely the same place (isoschizomers, 同切
點酶).
E. There are two major classes of restriction enzyme that differ in
where they cut the DNA, relative to the recognition site.
1. Type I restriction enzymes cut the DNA a long way from the
recognition sequence.
2. Type II restriction enzymes cut the DNA within the recognition
sequence. Some generate blunt ends, others give sticky ends.
Restriction fragment ends
Separation of DNA fragments by
gel electrophoresis
There are kinds of gels for electrophoresis:
A. agarose（瓊脂糖）and polyacrylamide
（聚丙烯醯胺）gel electrophoresis
(膠体電泳)
B. Agarose and polyacrylamide can act as
a molecular sieve
C. Agarose is better for separation of DNA
fragments >1 kb, and polyacrylamide
gel for < 1kb.
Electrophoresis can separate DNA
fragments from one another（電泳可將
DNA片段彼此分開）
DNA fragments are separated using an electrical
current:
a. Apparatus and material – casting tray, comb,
loading dye, ethidium bromide （溴化乙錠）and
power supply
b. DNA molecules have a negative electric charge
due to the phosphate groups which alternate
with sugar molecules to make up the backbone
of the DNA double helix.
c. Opposite electric charges tend to attract one
another.
Horizontal apparatus for
(agarose) gel electrophoresis
Gel electrophoresis
movement of
DNA in electrical
field
Gel electrophoresis for DNA
fragments
Pulsed-Field Gel Electrophoresis
(PFGE)
當兩個電磁場以規則的方式相
互交替，DNA分子在膠體中之淨
值移動仍然是從一端到另外一
端，移動方向或多或少還是直
線的。然而，隨著每一個電磁
場方向的改變，每一DNA分子在
繼續往前移動之前必須作90度
重新排列。這就是這種技術的
重點，因短的分子重新調整比
長的分子快，使得短的分子朝
膠體尾端前進的速度快。這種
新增的空間戲劇性地增大了膠
體的解析力，因此大至好幾千
kb 長的分子可以被分開。
Southern blot analysis
PCR reaction (聚合酵素鏈鎖反應)
PCR reaction (聚合酵素鏈鎖反應)
The Determining base sequence of
DNA fragments（定序列）
A. Maxam and Gilbert DNA sequencing
a. base modification chemicals:
1. DMS (dimethylsulfate)  G
2. DMS in the presence of formic acid  G and A
3. HZ (hydrazine)  T and C
4. HZ in the presence of NaCl  C
b. cleavage of modified base - piperidine
B. Sanger dideoxy - DNA sequencing
a. dideoxy dNTP
b. primers
C. Denaturing polyacrylamide sequencing gel
D. Autoradiography
E. Automated sequencing
Maxam & Gilbert Sequencing
Structure of an α-35Sdeoxynucleotide triphosphate
Dideoxynucleoside triphosphates
(ddNTP)
DNA sequencing with ddNTPs as
chain terminators
Profile of an automated
sequencing
Labelling the Probes
•Nick translation
•Random primimg
•3‘-end-labelling a DNA by end-filling
•End-labelling by polynucleotide kinase
Fig. 5.29
3‘-end-labelling a DNA by end-filling