III. Chromosome replication is coordinated with cell division

III. Chromosome replication isIII. Chromosome replication is coordinated with cell division coordinated with cell division

III. Chromosome replication isIII. Chromosome replication is coordinated with cell division 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 isIII. Chromosome replication is coordinated with cell division 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 isIII. Chromosome replication is coordinated with cell division 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 isIII. Chromosome replication is coordinated with cell division coordinated with cell division

IV. Some methods for DNA studyIV. Some methods for DNA study

Nucleic acids hybridize by base Nucleic acids hybridize by base pairingpairing

· A crucial property of double helix is the ability to separate the two · A crucial property of double helix is the ability to separate the two strands without disrupting covalent bond. This makes it possible for strands without disrupting covalent bond. This makes it possible for the strands to separate and reform under physiological conditions at thethe strands to separate and reform under physiological conditions at the ( very rapid) rates needed to sustain genetic functions.( very rapid) rates needed to sustain genetic functions.· Heating and some chemicals (e.g., NaOH) cause the two strands of a DNA · Heating and some chemicals (e.g., NaOH) cause the two strands of a DNA duplex to separate. duplex to separate. · Complementary single strands can renature when the temperature is · Complementary single strands can renature when the temperature is reduced. reduced. ·  Denaturation and renaturation/hybridization can occur with DNA-DNA, ·  Denaturation and renaturation/hybridization can occur with DNA-DNA, DNA-RNA, or RNA-RNA combinations, and can be intermolecular or DNA-RNA, or RNA-RNA combinations, and can be intermolecular or intramolecular. intramolecular. · The · The TTmm is the midpoint of the temperature range for denaturation. is the midpoint of the temperature range for denaturation.

· The ability of two single-stranded nucleic acid preparations · The ability of two single-stranded nucleic acid preparations to hybridize is a measure of their complementarity. to hybridize is a measure of their complementarity.

DNA MeltingDNA Melting

• 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

• 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

Denatured single strands of DNA can reDenatured single strands of DNA can renature to give the duplex form.nature to give the duplex form.

Filter hybrydizationFilter hybrydization

Calculation of Tm for a DNA Calculation of Tm for a DNA moleculemolecule

1.1.TmTm = (G+C)%/2.44 + 81.5 + 16.6log = (G+C)%/2.44 + 81.5 + 16.6log[Na+][Na+]

2. For oligonucleotide: 2. For oligonucleotide:

TmTm = 4 (G+C) + 3 (A+T) = 4 (G+C) + 3 (A+T)

3. In the presence of foramide: 3. In the presence of foramide:

minus minus 0.67 0.67 00C per %C per %

4. High stringency and low stringency4. High stringency and low stringency

Measurement of nucleic acid Measurement of nucleic acid concentrationconcentration

1. Hypochromic effect: The heterocyclic rings of 1. Hypochromic effect: The heterocyclic rings of

nucleotides absorb light strongly in UV range (with nucleotides absorb light strongly in UV range (with

maximum close to 260 nm that is characteristic for each base). maximum close to 260 nm that is characteristic for each base).

But the absorption of DNA itself is 40% less than would be But the absorption of DNA itself is 40% less than would be

displayed by a mixture of free nucleotides of the same displayed by a mixture of free nucleotides of the same

composition.composition.

2. Concentration of nucleic acid in a solution (ug/ml):2. Concentration of nucleic acid in a solution (ug/ml):

DNA: ODDNA: OD260260 X 50 X (dilution factor) X 50 X (dilution factor)

RNA: ODRNA: OD260260 X 40 X (dilution factor) X 40 X (dilution factor)

Oligo: ODOligo: OD260260 X 30 X (dilution factor) X 30 X (dilution factor)

Restriction enzymesRestriction enzymes

Restriction and modification( 限制與修飾 )

Restriction enzymesRestriction enzymesA. Cleave DNA at specific sequences (recognition A. Cleave DNA at specific sequences (recognition sequence, cutting site ).sequence, cutting site ). (( 一種可以針對特定一種可以針對特定 DNADNA 序列進行辨認與切割的蛋白質序列進行辨認與切割的蛋白質 ))B. Firstly discovered by Arber and Smith (late 1960s)B. Firstly discovered by Arber and Smith (late 1960s)C. Boyer (1969) first isolated C. Boyer (1969) first isolated EcoEcoRIRID. Different restriction enzymes may share the same D. Different restriction enzymes may share the same recognition sequence although they do not necessary recognition sequence although they do not necessary cut at precisely the same place (isoschizomers, cut at precisely the same place (isoschizomers, 同切同切 點酶點酶 ).).E. There are two major classes of restriction enzyme thE. There are two major classes of restriction enzyme th

at differ in where they cut the DNA, relative to the rat differ in where they cut the DNA, relative to the recognition site.ecognition site.

1. Type I restriction enzymes cut the DNA a long way 1. Type I restriction enzymes cut the DNA a long way from the from the

recognition sequence.recognition sequence. 2. Type II restriction enzymes cut the DNA within th2. Type II restriction enzymes cut the DNA within th

e recognition e recognition sequence. Some generate blunt ends, others give sequence. Some generate blunt ends, others give

sticky ends.sticky ends.

Restriction fragment endsRestriction fragment ends

Separation of DNA fragments by Separation of DNA fragments by gel electrophoresisgel electrophoresisThere 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 fragmElectrophoresis can separate DNA fragments from one anotherents from one another （電泳可將（電泳可將 DNADNA 片片段彼此分開）段彼此分開）DNA fragments are separated using an electrical DNA fragments are separated using an electrical current: current: a. Apparatus and material – casting tray, comb, a. Apparatus and material – casting tray, comb, loading dye, ethidium bromide loading dye, ethidium bromide （溴化乙錠）（溴化乙錠） and and power supplypower supply b. DNA molecules have a negative electric charge b. DNA molecules have a negative electric charge due to the phosphate groups which alternate due to the phosphate groups which alternate with sugar molecules to make up the backbone with sugar molecules to make up the backbone of the DNA double helix.of the DNA double helix. c. Opposite electric charges tend to attract one c. Opposite electric charges tend to attract one another.another.

Horizontal apparatus for (agarosHorizontal apparatus for (agarose) gel electrophoresise) gel electrophoresis

movement ofDNA in electricalfield

Gel electrophoresisGel electrophoresis

Gel electrophoresis for DNA Gel electrophoresis for DNA fragmentsfragments

Pulsed-Field Gel Electrophoresis Pulsed-Field Gel Electrophoresis (PFGE)(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 analysisSouthern blot analysis

PCR reaction PCR reaction (( 聚合酵素鏈鎖反聚合酵素鏈鎖反應應 ))

PCR reaction PCR reaction (( 聚合酵素鏈鎖反聚合酵素鏈鎖反應應 ))

The Determining base sequence of DNThe Determining base sequence of DNA fragmentsA fragments （定序列）（定序列）A. Maxam and Gilbert DNA sequencingA. Maxam and Gilbert DNA sequencing a. base modification chemicals:a. base modification chemicals: 1. DMS (dimethylsulfate) 1. DMS (dimethylsulfate) G G 2. DMS in the presence of formic acid 2. DMS in the presence of formic acid G and A G and A 3. HZ (hydrazine) 3. HZ (hydrazine) T and C T and C 4. HZ in the presence of NaCl 4. HZ in the presence of NaCl C C b. cleavage of modified base - piperidineb. cleavage of modified base - piperidineB. Sanger dideoxy - DNA sequencingB. Sanger dideoxy - DNA sequencing a. dideoxy dNTPa. dideoxy dNTP b. primersb. primersC. Denaturing polyacrylamide sequencing gelC. Denaturing polyacrylamide sequencing gelD. AutoradiographyD. AutoradiographyE. Automated sequencingE. Automated sequencing

Maxam & Gilbert Sequencing

Structure of an Structure of an α-α-3535S-deoxynuS-deoxynucleotide triphosphatecleotide triphosphate

Dideoxynucleoside triphosphates (dDideoxynucleoside triphosphates (ddNTP)dNTP)

DNA sequencing with ddNTPs as chaiDNA sequencing with ddNTPs as chain terminatorsn terminators

Profile of an automated Profile of an automated sequencingsequencing

Labelling the ProbesLabelling the Probes

•Nick translationNick translation•Random primimgRandom primimg•3‘-end-labelling a DNA by end-filling3‘-end-labelling a DNA by end-filling•End-labelling by polynucleotide kinaseEnd-labelling by polynucleotide kinase

Fig. 5.29

3‘-end-labelling a DNA by end-filling