main enzymes used in molecular biology (theory and practice) by jean-pierre herveg and a lot of...

Main enzymes usedin Molecular biology

(theory and practice)

by Jean-Pierre Herveg and a lot of friends atthe Brussels Branch of the Ludwig Institute for Cancer research (Licr) and the

Christian de Duve* Institute for cellular Patholgy (ICP).

April 2006Université Catholique de Louvain

Avenue E. Mounier, 1200 Brussels (Belgium)

----------------------*Christian de Duve got the Noble Prize in 1974. He and his team discovered both lysosomes and peroxysomes.

DNA Polymerases

Enzymes classification

(in fact this classification is more a classification of reactions than a classification of enzymes.There are 6 classes of reactions and thus six classes of enzymes.The DNA pols are in the EC2 goup, the second group.

EC 1 Oxidoreductases: catalyze oxidation/reduction reactions

EC 2 Transferases: transfer a functional group (a nucleotide)

EC 3 Hydrolases: catalyze the hydrolysis of various bonds EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation EC 5 Isomerases: catalyze isomerization: changes within a single molecule EC 6 Ligases: join two molecules creating covalent bonds

DNA pol is an EC 2 Transferase

Exemples:

EC 2.-.-.- transferases (any transfer).EC 2.7.-.- transferring phosphorous-containing groups. EC 2.7.7.- transferring nucleotides: nucleotidyltransferases.

EC 2.7.7.7 DNA-directed DNA pol.

EC 2.7.7.49 RNA-directed Dna pol.

A reverse transcriptase is an RNA- directed DNA polEC 2.7.7.49. This enzyme is in the EC2.7.7. Group

____________________Question:what is the difference between DNA pol and RT-DNA pol ?

How does the EC classification work ?

DNA PolymerasesEnzymes that replicate DNA using a DNA or RNA templates*.

DNA-directed DNA polymerase EC 2.7.7.7RNA-directed DNA polymerase (Reverse transcriptase) EC 2.7.7.49

Most organisms have more than one type of DNA polymerase(for example, E. coli has five DNA polymerases), but all work using the same 4 basic rules.

Polymerization

1. requires a template (DNA or RNA) to copy the complementary strand. 2. requires a pre-existing primer (DNA or RNA) from which to extend 5’ to 3’.

3. occurs only in the 5' to 3’ direction4. requires 4 dNTPs: dATP, dGTP, dCTP, dTTPhowever, the reaction works also with ddNTPs even when they are bound to

large molecules like fluorochromes etc.In this case, the size of catalytic core of the DNA pol had to be increased)

For replication, in most organism, the primer is a RNA molecule(in some viruses the primer is a protein).

Bacteria have 5 known DNA polymerases:

* Pol I: Is implicated in DNA repairhas both 5'->3' and3'->5' exonuclease activity.

* Pol II: Pol II is involved in replication of damaged DNAhas a 3'->5' exonuclease activity (proof reading).

* Pol III: is the main polymerase in bacteria (elongates in DNA replication)as such it has 3'->5' exonuclease proofreading ability.

* Pol IV: is a Y-family DNA polymerase * Pol V: is a Y-family DNA polymerase and participates in bypassing DNA damage

E. coli DNA Polymerase IDNA Polymerase I from E. coli was the first DNA polymerase characterized.

The enzyme is a single large protein with a molecular weight of approximately 103 kDaThe enzyme requires a divalent cation (Mg++) for activity

It has 3 enzymatic activities:

1. 5'-to-3' DNA Pol activity 2. 3'-to-5' exonuclease (Proofreading activity) 3. 5'-to-3' exonuclease (Nick translation or Taqman activity)

The rate of DNA synthesis by pol is only 20 nucleotides/second (1200 nt/minutes)much slower than the rate of 1,000 nucleotides/second measured for the replication of E. coli DNA.

DNA polI is not the main enzyme used to replicate DNA.

DNA polymerase I removes the RNA primer from the lagging strand and fills in the necessarynucleotides.

--------------------------questions:1. What is the lagging strand ?


1. 5'-to-3' DNA Pol activity 2. 3'-to-5' exonuclease (Proofreading activity) 3. 5'-to-3' exonuclease (Nick translation or Taqman activity)

-------------------------------------Questions:

1. Cites the activities of E. Coli DNA polymerase I.2. What is the rate of DNA synthesis of the E. Coli DNA polymerase I.3. What does “proofreading activity means”?4. What do “nick translation and Taqman activity mean”?5. What is the rate of DNA synthesis by E. Coli DNA pol ?


1. 5’ to 3' DNA Pol activity

5‘ tatctggttgatcctgcc 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttgatcctgccagtattatatgctgaattcag 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

2. 3’ to 5' exonuclease (Proofreading activity)

5‘ tatctggttgatcctgccagtagtatatgctaaaatcagagattaaggcatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘


3. 5’ to 3' exonuclease (Nick translation and Taqman activity)

5‘ tatctggttgatcctgcc tgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttgatcctgcc ttaagccatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

DNA polymerase III in the replisomeis the primary enzyme complex involved in prokaryotic DNA replication.

The complex has high processivity (i.e. the number of nucleotides added per binding event) and,specifically referring to the replication of the E.coli genome,works in conjunction with four other DNA polymerases (Pol I, Pol II, Pol IV, and Pol V).Being the primary holoenzyme involved in replication activity, the DNA Pol III holoenzymealso has proofreading capabilities that correct replication mistakes by means ofexonuclease activity working 3'->5'.

DNA Pol III is a component of the replisome, which is located at the replication fork.

The replisome is composed of the following:

2 DNA Pol III enzymes, made up of α, ε, θ subunits.the α subunit has polymerization activity.the ε subunit has proofreading activity.the θ subunit stimulates the ε subunit's proofreading.

2 β units which act as sliding DNA clamps, they keep the polymerase binded to the DNA.It has two subunits.

2 τ units which connect the 2 DNA Pol III enzymes. 1 γ unit which acts as a clamp loader for the lagging strand Okazaki fragments,

helping the two β subunits to form a unit and bind to DNA.The γ unit is made up of 5 γ subunits.

-------------------------------Question:1. What is processivity ?2. What’s an Okazaki fragment ?

Thermoresistant DNA pol

Thermoresistant DNA pols are used in PCR. Thermoresistant DNA pols exist in extremophileBacteria and Archaea. Extremophile means living in extreme condtions. Some Bacteria andArchaea are living in hot geyser or in the bottoms of ocean, near erupting volcanoes. Their DNA pol resists then to high temperature.

They are used at 68 to 72° C. At this temperature, the primers are annealed to perfectlyComplementary sequences. In addition we don’t need to add DNA pols after each PCR cycleAs it was the case with non thermoresistant DNA pols.

The qualities of these DNA pols is expressed by the time they can be incubated at 94° C beforeShowing a decrease of half or their activity.

Bacterial DNA polsThey don’t have a proof reading activity. This means that they don’t have a 3’to 5’ endonucleaseActivity. On example is the Taq DNA pol. The gene coding this enzyme is prepared fromThermus aquaticus. Taq DNA pol incorporates 20 nt per second at their optimum pH andtemperature

Archaeal DNA polsThey have a proof reading activity, but they are 10 times less active. Pfu is an example ofthis kind of DNA pol.

---------------------------------question:Why is Pfu DNA pol 10 times less active than Taq DNA pol ?

Home made thermoresistant DNA pols.

Instead of giving their money to rich international companies, many laboratories prepare theirown thermoresistant DNA pol.

They have a strain of E. coli, transformed by a plamid coding such an enzymze.The protein which is expressed is thermoresistant. Then, the broth in which this bacteria was grownis heated for one hour at 70° C. The only protein which resist is the thermoresistant DNA pol.

This heated solution can be used directly as an enzyme.

The heated preparation could also be further purified by passing it through a resin.This resin is expensive.

---------------------------QuestionHow can we prepare a “home made” thermoresistant DNA pol ?

type II restriction enzyme: EC 3.1.21.4

These Enzymes are hydrolases that hydrolase ester bonds:

EC 1 Oxidoreductases: catalyze oxidation/reduction reactions EC 2 Transferases: transfer a functional group EC 3 Hydrolases: catalyze the hydrolysis of various bonds EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation EC 5 Isomerases: catalyze isomerization changes within a single molecule EC 6 Ligases: join two molecules with covalent bonds

EC3.1.

E.C.3.1.-.- Acting on ester bonds. [ 1076 PDB entries ]E.C.3.2.-.- Glycosylases. [ 1465 PDB entries ]E.C.3.3.-.- Acting on ether bonds. [ 16 PDB entries ]E.C.3.4.-.- Acting on peptide bonds (peptide hydrolases). [ 1582 PDB entries ]E.C.3.5.-.- Acting on carbon-nitrogen bonds, other than peptide bonds. [ 306 PDB entries ]E.C.3.6.-.- Acting on acid anhydrides. [ 153 PDB entries ]E.C.3.7.-.- Acting on carbon-carbon bonds. [ 8 PDB entries ]E.C.3.8.-.- Acting on halide bonds. [ 31 PDB entries ]E.C.3.9.-.- Acting on phosphorus-nitrogen bonds. [-]E.C.3.10.-.- Acting on sulfur-nitrogen bonds. [-]E.C.3.11.-.- Acting on carbon-phosphorus bonds. [-]E.C.3.12.-.- Acting on sulfur-sulfur bonds. [-]E.C.3.13.-.- Acting on carbon-sulfur bonds. [-]

Restriction enzymes

EC 3.1.21.4

Common name: type II site-specific deoxyribonucleaseOther name: type II restriction enzyme

Reaction: Endonucleolytic cleavage of DNA giving specific double-stranded fragmentswith terminal 5'-phosphates

These enzymes recognize specific short DNA sequences (palindrome) and cleavewthin the palindrome. Palindromic sequences are called restriction sites

Example:gaattc g aattccttaag cttaa g

The fragments created by a restriction endonuclease possess either a restiction siteat both ends, or at one end only, depending of their position on the dsDNA molecule:

Restriction sites are palindromic element:

What is a palindrome ?A palindrome is a word, phrase, verse, or sentence that reads the same backward or forward.

Exemple with words: a nut for a jar of tuna.

Exemple with a restriction site:

The site recognised by Eco RI is

gaattccttaag

The site recognised by Hae III is

ggccccgg

Each time these enzymes recognize their specific sites, they bind to it and cleavewithin it.

-------------------------------Question:1. What’s a palindrome in molecular biology, give an example of a palindrome ?

Palindrome



a SNP within a palindrome


5‘ tatctggttgatcctgccagtattatatgctgtattcagagattaagccatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacataagtctctaattcggtacgtacacat 5‘

-----------------------question:1. What‘s a SNP within a palindrome.2. What‘s a RFLP ?

Each time these enzymes recognize their specific sites, they bind to it and cleavewithin it, giving restriction fragments

example with Eco RI (sticky ends):

5‘ tatctggttgaattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaacttaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttg 3‘3‘ atagaccaacttaa 5‘

+5‘ aattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ gcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

example with Hae III (blunt ends):

5‘ tatctggttggccttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaaccggaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttgg 3‘3‘ atagaccaacc 5‘

+5‘ ccttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ ggaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

Restriction sites: cohesive and blunt ends

1. Cohesive ends (sticky ends)extremos pegajosos o cohesivos

5’ protrusion (5’ saliente) (Eco RI):

5‘ tatctggttgaattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaacttaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttg 3‘3‘ atagaccaacttaa 5‘

+5‘ aattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ gcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

3’ protrusion (3’ saliente) (Pst I):

5‘ tatctggttctgcaggccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaagacgtccggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttctgca 3‘3‘ atagaccaag 5‘

+5‘ ggccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ acgtccggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

1. Blunt endsextremos romos

example: Pvu II

5‘ tatctggttcagctgttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaagtcgacaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ tatctggttcag 3‘3‘ atagaccaagtc 5‘

+

5‘ ctgttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ gacaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

The Klenow fragment of DNA pol I:Filling or suppressing the protruding ends

the Klenow fragment has two acitivities:5’ --> 3’ DNA pol activity5’ --> 3’ exonuclease activity

1. filling a 5’ protruding end using the DNA pol activity.The molecule could be labeled if t is either radioactive or fluorescent

5' tatctggttg 3’ 3' atagaccaacttaa 5’

------>

5' tatctggttgaatt 3’ 3' atagaccaacttaa 5’

2. suppressing a 3’ protruding end using the exonuclease activity.

5‘ tatctggttctgca 3‘3‘ atagaccaag 5‘

------>

5‘ tatctggttc 3‘3‘ atagaccaag 5‘ + t, g, c and a

Frequency of restriction sites (sf):

1/4N

N = number of bases in the site4 means that in DNA we have 4 nucleotides (A,G, T and C)

Examples:

1. Eco RI, N = 6, sf = 1/46 = 1/4096This means that we could encounter one site every 4096 nucleotide (nt).

2. Hae III, N = 6, sf = 1/44 = 1/256This means that we could encounter one site every 256 nucleotide (nt).

5‘ tatctggttggccttcgccagtattggatcctgaattcagagattaagccatgcatgtgta 3‘ 3‘ atagaccaaccggaagcggtcataacctaggacttaagtctctaattcggtacgtacacat 5‘

Asp 718 (5’ protruding)5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5‘

Kpn I (3’ protruding) 5‘ tatctggttggccttcgccagtattggatc ctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaaccggaagcggtcataac ctaggacttaagtctctaattcggtacgtacacat 5‘

Sau 3AI (5’ protruding)5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5‘

Bam HI (5’ protruding) 5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5‘

but

Sau 3AI5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5

Sau 3AI5‘ tatctggttggccttcgccagtatta gatcgtgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaaccggaagcggtcataatctag cacttaagtctctaattcggtacgtacacat 5

T 7 Promoter Eco RI Not I T3 promter5’ ttaatacgactcactataggctagcctcgagaattcacgcgtggtacctctagagtcgacccgggcggccgcttccctttagtgagggttaatg 3’.

example: a mammalian vector: why a mammalian ?

----------------------------Question:What’s a cloning sites

DNA Ligase (EC 6)DNA ligases catalyze formation of a phosphodiester bond between the 5' phosphateof one strand of dsDNA and the 3' hydroxyl of an other.

This enzyme is used to covalently link or ligate fragments of dsDNA together.Most commonly, the reaction involves ligating a fragment of DNA into a plasmid vector, which is a fundamental technique in recombinant DNA work.A DNA ligase repair the cut made by a restriction endonuclease.

The most widely used DNA ligase is derived from the T4 bacteriophage.T4 DNA ligase requires ATP as a cofactor. EC 6.5.1.1 Common name: T4 DNA ligase (ATP)

A DNA ligase from E. coli is also available, but is not commonly used.it uses NAD as a cofactor. EC 6.5.1.2Common name: DNA ligase (NAD+)

-----------------------------Questions:1. In T4 DNA ligase, what does T4 means ?2. What is the cofactor used by T4 DNA ligase ?

A ligase is an enzyme that catalyses the joining of two moleculesby forming a new chemical bond, with accompanying hydrolysis ofATP or similar molecules.

A DNA ligase is an enzyme that catalyze the joining of two DNA moleculesby forming a phosphodiester bond between the 5' phosphate of one strand of DNAand the 3' hydroxyl of the another.with the accompanying hydrolysis of ATP (EC 6.5.1.1) or NAD (EC 6.5.1.2).

Among all the enzyme reactions, the ligase reaction is classifiedas EC 6 in the EC number classification of enzyme reactions:

EC 1 Oxidoreductions. oxydoreductasesEC 2 Transferts: transferases transfer a functional group (a methyl or phosphate group)EC 3 Hydrolysis: hydrolases catalyze the hydrolysis of various bondsEC 4 Lyase reactions: Lyases cleave various bonds by means other than hydrolysisand oxidationEC 5 Isomerisations: isomerases catalyze isomerization changes within a single moleculeEC 6 Ligation: ligases join two molecules with covalent bonds

The common names of ligases sometimes include the word "ligase," such as”DNA ligase”, an enzyme joining DNA fragments.But, ligase could be absent of the name”Synthetase" (when ligases are used to synthesize new molecules”Carboxylase" (when ligases are used to add carbon dioxide to a molecule).

Classification.Ligases are classified into six subclasses:EC 6.1 includes ligases used to form carbon-oxygen bonds: acylates a tRNA with its aa aa-tRNA ligasesEC 6.2 includes ligases used to form carbon-sulfur bonds: synthesizes acyl-CoA derivatives.EC 6.3 includes ligases used to form carbon-nitrogen bonds: amide synthasesEC 6.4 includes ligases used to form carbon-carbon bonds: carboxylating enzymes,mostly biotinyl-proteinsEC 6.5 includes ligases used to form phosphoric ester bonds, DNA ligase = EC 6.5.1.1 and EC 6.5.1.2EC 6.6 includes ligases used to form nitrogen-metal bonds

------------------------------Question:Explain the reaction catalyzed by T4 DNA ligase and ATP.

A ligation reaction requires 3 reagents :

At least 2 fragments of DNA With either blunt or compatible cohesive ("sticky") ends.

A buffer which contains ATP.The buffer is usually provided or prepared as a 10X concentrate which,after dilution, yields an ATP concentration of roughly 0.25 to 1 mM.Most restriction enzyme buffers will work if supplemented with ATP.

T4 DNA ligase. A typical reaction for inserting a fragment into a plasmid vectorwould utilize about

0.01 (sticky ends) to 1.0 (blunt ends) units of ligase.

Intermolecular ligation:(liagación intermolecular)

1. With cohesive ends (sticky ends, here Eco RI gaattc/cttaag):

5‘ tatctggttgatcctgccaattattatatgctgOH 3‘ atagaccaactaggacgttcataatatacgaccttaaPOH

+ OHPaattcagagattaagccatgcatgtgta 3‘

HOgtctctaattcggtacgtacacat 5‘

+ T4 DNA ligase + ATP 5‘ tatctggttgatcctgccaattattatatgctgaattcagagattaagccatgcatgtgta 3‘ 3‘ atagaccaactaggacgttcataatatacgaccttaagtctctaattcggtacgtacacat 5‘

2. With bunt ends

5‘ tatctggttgatcctgccaattattatatgctOH 3‘ atagaccaactaggacgttcataatatacgacPOH

+ OHPagagattaagccatgcatgtgta 3‘

HOtctctaattcggtacgtacacat 5‘

+ T4 DNA ligase + ATP 5‘ tatctggttgatcctgccaattattatatgctagagattaagccatgcatgtgta 3‘ 3‘ atagaccaactaggacgttcataatatacgactctctaattcggtacgtacacat 5‘

Intramolecular ligation:(liagación intramolecular)

Phage

Phage (fagos )

Cos LCos R

+ T4 DNA ligase + ATP

cut and paste:

1. Cut the DNA to be inserted, using Eco RI and Not I:

5‘ tatctggttgagaattctcctgccagtagtatatgctaaaatcagaggcggccgcattaaggcatgcatgtgta 3‘3‘ atagaccaactcttaagaggacggtcataatatacgacttaagtctccgccggcgtaattcggtacgtacacat 5‘

Eco RI Not I5‘ tatctggttgagaattctcctgccagtagtatatgctaaaatcagaggcggccgcattaaggcatgcatgtgta 3‘3‘ atagaccaactcttaagaggacggtcataatatacgacttaagtctccgccggcgtaattcggtacgtacacat 5‘

+ Eco RI 5‘ tatctggttgag aattctcctgccagtagtatatgctaaaatcagaggcggccgcattaaggcatgcatgtgta 3‘3‘ atagaccaactcttaa gaggacggtcataatatacgacttaagtctccgccggcgtaattcggtacgtacacat 5‘

+ Not I 5‘ tatctggttgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcattaaggcatgcatgtgta 3‘3‘ atagaccaactcttaa gaggacggtcataatatacgacttaagtctccgccgg cgtaattcggtacgtacacat 5‘

fragment ready to be pasted:

5‘aattctcctgccagtagtatatgctaaaatcagaggc 3‘ 3‘ gaggacggtcataatatacgacttaagtctccgccgg 5‘

5‘ tatctggttgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcattaaggcatgcatgtgta 3‘3‘ atagaccaactcttaa gaggacggtcataatatacgacttaagtctccgccgg cgtaattcggtacgtacacat 5‘

------------------------------------question:explain The „cut and paste“ reaction in molecular biology.

2. Cut the vector, using Eco RI and Not I:

example: our mammalian vector

--------------------Question:1. Why are there T4 and SP6 promoter elements at both ends of the cloning site ?2. What’s a MCS ?3. Why is there an intron before the cloning site in this vector ?

T 7 Promoter Eco RI Not I T3 promter5’ ttaatacgactcactataggctagcctcgagaattcacgcgtggtacctctagagtcgacccgggcggccgcttccctttagtgagggttaatg 3’.

5’ …agcctcgagaattcacgcgtggtacctctagagtcgacccgggcggccgcttcccttta… 3’3’ …tcggagctcttaagtgcgcaccatggagatctcagctgggcccgccggcgaagggaaat… 5’

5’ …agcctcgag aattcacgcgtggtacctctagagtcgacccgggc ggccgcttcccttta… 3’3’ …tcggagctcttaa gtgcgcaccatggagatctcagctgggcccgccgg cgaagggaaat… 5’



2. Paste DNA into the vector using Ligase + ATP

fragment to be pasted

aaaaaaaaaaaa5‘ aattctcctgccagtagtatatgctaaaatcagaggc 3‘ aaaaaaaaaaaa3‘aaaaagaggacggtcataatatacgacttaagtctccgccgg 5‘

open vector5’ …agcctcgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcttcccttta… 3’3’ …tcggagctcttaag aggacggtcataatatacgacttaagtctccgccgg cgaagggaaat… 5’

5’ …agcctcgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcttcccttta… 3’3’ …tcggagctcttaag aggacggtcataatatacgacttaagtctccgccgg cgaagggaaat… 5’

+ ligase and ATP

5’ …agcctcgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcttcccttta… 3’3’ …tcggagctcttaag aggacggtcataatatacgacttaagtctccgccgg cgaagggaaat… 5’

Isoschizomers: enzymes that recognize the same siteAsp 718 5’ g gatcc 3’ 3’ cctag g 5’ Kpn I 5’ ggatc c 3’ 3’ c ctagg 5’ Bam HI 5’ g gatcc 3’ 3’ cctag g 5’

Enzyme’s family: enzymes producing the same endings

gatc: Sau 3AI, Bgl I, Bam HI, Bcl II, Xho IIctag: Mae I, Spe I, Nhe I, Avr I, Xba I Bam HI 5’ g gatcc 3’ 3’ cctag g 5’Sau 3AI 5’ -gatc 3’ 3’ ctag- 5’

Enzymes:


EC 2 Transferases: transfer a functional group

EC 3 Hydrolases: catalyze the hydrolysis of various bonds EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation EC 5 Isomerases: catalyze isomerization changes within a single molecule EC 6 Ligases: join two molecules with covalent bonds

Exemples:

EC 2.-.-.- Transferases.EC 2.7.-.- Transferring phosphorous-containing groups. EC 2.7.7.- Nucleotidyltransferases.EC 2.7.7.6. DNA-dependent RNA polymerase II EC 2.7.7.7 DNA-directed Dna polymerase.

------------------------------Question:1. What does “DNA dependant” means in DNA dependant RNA pol ?2. Does the RNA pol need a primer ?

DNA dependent RNA polymerases (RNA pol)

T7 RNA Polymerase

a DNA-dependent RNA polymerase from a bacteriophage

T7 RNA Polymerase is a DNA-dependent RNA polymerase that exhibits extremely high specificityfor its cognate promoter sequences: 5’ tgtaatacgactcactataggg 3’ (In red, the promotion start site)

Only DNA cloned downstream from a T7 promoter can serve as a templatefor T7 RNA Polymerase-directed RNA synthesis.

It is generally provided (10-20 u /µl) with another tube containing the concentrated (5x or 10 x) reaction buffer you should use.

It is used in molecular biology for the production of:

1. RNA templates for in vitro translation.2. probes for nucleic acid hybridizations (microarrays).3. RNA processing substrates.4. antisense RNA.

Other phage RNA pol available

SP6 RNA pol, T3 RNA pol,They are produced by recombinant technology.

---------------------------QuestionsWhat are the uses of T7 RNA pol.

T7 promoter Eco RI Bam HI5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcct

Hind III SP6 promoterctagagtcgacctgcaggcatgcaagcttgagtattctatagtgtcacctaaat 3’

DNA primase ( in replication) is a DNA dependent RNA pol

DNA Primase and replication:

5‘ ugguugauccugcc 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ ugguugauccugccagtattatatgctgaattcagagattaagccatgcat---> 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

(AMV = Avian Myeloblastosis Virus)

Enzymes:


EC 2 Transferases: transfer a functional group

EC 3 Hydrolases: catalyze the hydrolysis of various bonds EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation EC 5 Isomerases: catalyze isomerization changes within a single molecule EC 6 Ligases: join two molecules with covalent bonds Exemples:

EC 2.-.-.- Transferases.EC 2.7.-.- Transferring phosphorous-containing groups. EC 2.7.7.- Nucleotidyltransferases. EC 2.7.7.7 DNA-directed Dna polymerase.

EC 2.7.7.49 RNA-directed DNA polymerase M-MLV RT DNA pol

AMV reverse transcriptase

AMV RT DNA pol (AMV RT DNA pol)

Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT)catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybridThis enzyme is DNA/RNA dependent !

It requires1. primer (DNA primers are more efficient than RNA primers)2. Mg2+ or Mn2+ ions.

The enzyme possesses an intrinsic RNase H activity.Both nonionic detergents and sulfhydryl compounds stabilize the enzyme activity in vitro.AMV RT is the preferred reverse transcriptase for templates with high secondary structuredue to its stability at higher reaction temperatures (37–58°C).

It is generally provided (5-10 u /µl) with another tube containing the concentrated (5x or 10 x) reaction buffer you should use.

Applications

First- and second-strand synthesis of cDNA.Primer extensions.RT-PCR. Up to 10 µl of an RT reaction containing AMV RT and the supplied AMV RT Reaction Buffercan be added to a 50 µl PCR amplification reaction that uses Taq DNA Polymerase (20 %).------------------------Question:What’s a RNA high secondary structure.

M-MLV RT DNA pol (reverse transcriptase)(EC 2.7.7.49 RNA-directed DNA polymerase)

Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is an RNA-dependent DNA polymerase that can be used in cDNA synthesiswith long messenger RNA templates (>5kb).The enzyme is a product of the pol gene of M-MLV: it consists of a single subunitwith a molecular weight of 71kDa.The RNase H activity of M-MLV RT is weaker than the commonlyused Avian Myeloblastosis Virus (AMV) reverse transcriptase.

ApplicationsFirst-strand synthesis of cDNA.Primer extensions.

RT-PCR: Up to 10 µl of an RT reaction containing AMV RT and the suppliedAMV RT Reaction Buffer can be added to a 50 µl PCR amplification reaction that usesTaq DNA Polymerase.

T4 Polynucleotide Kinase catalyzes the transfer of the -phosphate from ATP to the5´-terminus of polynucleotides or to mononucleotides bearing a 5´-OH group.

The enzyme is used to end phosphorylate RNA, DNA and synthetic oligonucleotidesprior to subsequent manipulations such as ligation.

The DNA 5´ End-Labeling System is a complete system for phosphorylating bothdouble- and single-stranded DNA and RNA with T4 Polynucleotide Kinase and [-32P] ATP.The system includes enzymes, buffers and control DNA standards to measure reaction efficiencies.Calf or Chrimp Intestinal Alkaline Phosphatase is included for removal of the 5´-phosphateprior to labeling with T4 Polynucleotide Kinase

applications

1. 5´ end-labeling of single- or double-stranded DNA and RNA molecules for use as probes,for sequencing or for DNA-protein footprinting.

2. 2. Phosphorylation of DNA prior to cloning.

T4 Polynucleotide Kinase

1. An alcaline phosphatase cleave off the 5’ phosphates

5‘P tatctggttgaattcagagattaagccatgcatgtgta 3‘ 3‘ atagaccaacttaagtctctaattcggtacgtacacat P 5‘

+ alcaline phosphatase

5‘ tatctggttgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaacttaagtctctaattcggtacgtacacat 5‘

1. Polynucleotide kinase replaces this phosphate by the labeleed phosphate ofa radioactive phosphate from a radioactive ATP (a gamma phosphate!)

5‘P tatctggttgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaacttaagtctctaattcggtacgtacacat P 5‘

ATPhttp://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookATP.html

Probe labelling

Among the various methods of labelling a probe we will describeThe following ones:1. Random priming2. Nick translation3. Polynucleotid kinase

Random priming Random primers:

5‘ ctggtt5‘ gccagt.........

One labelled nucleotide, t

5‘ctggtt3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘ctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘gccagt 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

5‘gccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

Nick translation

Using DNAase, we made a few number of nick (here in red)


The 5‘ --> 3‘ exonucleasic activity of DNA pol I makes a gap fom a nick

5‘ tatctggttgatc----------tatatgctgaattcagagattaagccatgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

The DNA pol activity of DNA pol I uses the left sequence as a primer

5‘ tatctggttgatcctgccagtattatatgctgaattcag---------tgcatgtgta 3‘3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘

Alkaline Phosphatase from the Calf Intestine (CIAP),catalyzes the hydrolysis of 5´-phosphate groups from DNA, RNA,and ribo- and deoxyribonucleoside triphosphates.

This enzyme is used

1. to prevent recircularization and religation of linearized cloning vector DN by removing phosphate groups from both 5´-termini

2. for the dephosphorylation of 5´ phosphorylated ends of DNA or RNAfor subsequent labeling with [32P]ATP and T4 Polynucleotide Kinase.CIAP is active on 5´ overhangs, 5´ recessed and blunt ends.

Alkaline Phosphatase from the Calf Intestine (CIAP)

produced by the fungus Tritirachium album Limber, is a serine protease.It exhibits a very broad cleavage specificity. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples . It has been purified to be free of RNase and Dnase activities.

The stability of Proteinase K in urea and SDS and its ability to digest native proteinsmake it useful for a variety of applications, including preparation of chromosomal DNAfor pulsed-field gel electrophoresis (2), protein fingerprinting (3,4) and removal of nucleases

Proteinase K

topoisomeraseA class of enzymes that alter the supercoiling of double-stranded DNA.(In supercoiling the DNA molecule coils up like a telephone cord, which shortens the molecule.)The topoisomerases act by transiently cutting one or both strands of the DNA.

Topoisomerase type I (EC 5.99.1.2 ) cuts one strandTopoisomerase type II (EC 5.99.1.3) cuts both strands of the DNAto relax the coil and extend the DNA molecule..

Aside from topoisomerases I and II, there are more discovered topoisomerases.Topoisomerase III may regulate recombination while topoisomerase IV regulatesthe process of segregating newly replicated chromosomes from one another.

Drugs

Many drugs operate through interference with the topoisomerases.The broad-spectrum fluoroquinolone antibiotics act by disrupting the function ofbacterial type II topoisomerases.Some chemotherapy drugs work by interfering with topoisomerasesin cancer cells:type 1 is inhibited by irinotecan and topotecan type 2 is inhibited by etoposide and teniposide.

1. DNA Transciption and replication:

The regulation of DNA supercoiling is essential to DNA transcription and replication,when the DNA helix must unwind to permit the proper function of the enzymatic machineryinvolved in these processes.Topoisomerases serve to maintain both the transcription and replication of DNA.

2. Negative supercoiling

All the naturally occuring double stranded DNAs are negatively supercoiled.

(1) Negative supercoiling is important for packing of long DNA molecules into highly restricted spacessuch as viral cells or chromosomal structures in nucleus because supercoils generate compact structures.For example:Length of a human chromosome is of the order of centimeters,the condensed chromosome that contain this DNA only a few nanometers long.If DNA is constrained to be linear, it would not fit into a cell.

(2) Negative supercoiling facilitates the DNA-strand separation during replication,transcription and recombination of DNA.

DNA supercoiling is regulated in every cell that influences many aspects of DNA metabolism.The normal biological functioning of DNA occurs only if it is in the proper topological state.

SUPERHELIX TOPOLOGY

In circular double helical DNA (as in bacteria), both strands are covalently joined to form a circularduplex molecule. A geometric property of such an assembly is that its number of coilscannot be changed, without first cleaving at least one of its strands. Two strands are said to betopologically bonded to each other because they cannot be separated without breaking covalent bonds.

The conformation of circular duplex DNA can be characterized by 3 parameters.(1)Linking number:L(2)Twist:T(3)Writhing number: W

These parameters are related by the equation:

L= T+W (http://education.vsnl.com/kedar/)

(1) LL is the number of times that one DNA strand winds about the other strand.L is constant in an intact circular duplex DNA. L can be changed only if a covalent linkagein DNA backbone is broken and reformed.

(2) TT is the number of complete revolutions that one strand makes around the duplex axis.In a particular conformation "T" is positive for right handed duplex turns.For B-DNA the T equals the number of base pairs divided by 10.5 (10.5 base pairs /helical turn).

(3) Wis the number of turns that duplex axis makes about the superhelix axis in the conformation.It is the measure of DNA superhelicity.For a relaxed circular duplex DNA, W = 0 , Therefore L = T. Hence for relaxed duplex DNA L issimply the number of base pairs /10.5 i. e. one linking number for 10.5 base pairs. Two ways of introducing one supercoil in a DNA with 10 duplex turns. The two closed circular formsare topologically equivalent i. e. they are interconvertible without breaking any covalent bonds.The two DNA conformations have same linking number "L" but differ in their twists and writhing number.

topoisomerases and supercoilIn a "relaxed" double-helical segment of DNA,the two strands twist around the helical axis once every 10.4 base pairs of sequence.To add or subtract twists (vueltas), as some enzymes can do, is to impose a strain (tensión).This strain is positive (adding twists) or negative (substrating twists).

If a DNA segment under twist strain (+or -) were to be closed into a circle by joining its two endsand then allowed to move freely, the circular DNA would contort into the shape of an 8,

DNA topoisomerases type I (EC 5.99.1.2 ) Type I topoisomerases function by nicking one of the strands of the DNA double helix twisting it around the other strand, and religating the nicked strand. Topoisomerases I changethe linking number in steps of 1. They pass a single DNA strand through a nick

This process doesn’t need energy.The torque (par de torsión)present in the DNA drives the uncoiling.

Type IA topoisomerases change the linking number of a circular DNA strand by units of strictly 1Type IB topoisomerases change the linking number by multiples of 1.

--------------------------Question:How does topoisomerase I and II work ?

DNA topoisomerases type II (EC 5.99.1.3 ) Type II topoisomerases cut both strands of the DNA helix simultaneously.Topoisomerases II change the linking number (L + T + W) in steps of 2 by passing both strands ofdouble-stranded DNA through a break.

Once cut, the ends of the DNA are separated, and a second DNA duplex is passed through the break.Following passage, the cut DNA is resealed.This reaction allows type II topoisomerases to change the linking number of a DNA loop by +/-2,and promotes chromosome disentanglement.

For example, DNA gyrase, a type II isomerase observed in E. coli and most other prokaryotes,introduces negative supercoils and decreases the linking number by 2. Gyrase also is able toremove knots from the bacterial chromosome.

Get the E. dispar rRNA, DNA sequence Z49256

ncbi: national center for biotechnology informationsnlm: national library of medicinenih: national institutes od healthgov: US governmentLearn how tu use PubMd: http://www.nlm.nih.gov/bsd/disted/pubmed.html

SSU: Small subunitEDISSSURR (1212896): E. Dipar SSU rRNAaccession number: Z49256

--------------------------search for: buscaraccession: nf adquisiciónrecord (nombre): (aquí) documento, archivo

LOCUS EDISSSURR 1949 bp DNA linear INV 01-MAR-1996DEFINITION E.dispar gene for small subunit ribosomal RNA.ACCESSION Z49256VERSION Z49256.1 GI:1212896KEYWORDS small subunit ribosomal RNA.SOURCE Entamoeba dispar ORGANISM Entamoeba dispar Eukaryota; Entamoebidae; Entamoeba.REFERENCE 1 (bases 1 to 1949) AUTHORS Novati,S., Sironi,M., Granata,S., Scaglia,M. and Bandi,C. TITLE Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the desing of primers for rapid differentiation from Entamoeba histolytica JOURNAL UnpublishedREFERENCE 2 (bases 1 to 1949) AUTHORS Bandi,C. TITLE Direct Submission JOURNAL Submitted (12-MAY-1995) Claudio Bandi, Ist. di Patologia Generale Veterinaria, Universita', di Milano, Via Celoria 10, Milano, 20133, ItalyFEATURES Location/Qualifiers source 1..1949 /organism="Entamoeba dispar" /mol_type="genomic DNA" /db_xref="taxon:46681" rRNA 1..1949 /product="small subunit ribosomal RNA"

ORIGIN 1 tatctggttg atcctgccag tattatatgc tgatgttaga gattaagcca tgcatgtgta 61 agtataaaga ccaagtagga tgaaactgcg gacggctcat tataacagta atagtttctt 121 tggttagtaa agtacaagga tagctttgtg aatgataaag ataatacttg agacgatcca 181 atttgtatta gtacaaagtg gccaatttat gtaagtaaat tgagaaatga cattctaagt 241 gagttaggat gccacgacaa ttgtagaaca cacagtgttt aacaagtaac caatgagaat 301 ttctgatcta tcaatcagtt ggtagtatcg aggactacca agattataac ggataacgag 361 gaattggggt tcgacatcgg agagggagct ttacagatgg ctaccacttc taaggaaggc 421 agcaggcgcg taaattaccc actttcgaat tgaagaggta gtgacgacac ataactctag 481 agttgagtaa aatcaattct tgaaggaatg agtaggaggt aaattctcct acgaaatcaa 541 ttggagggca agtctggtgc cagcagccgc ggtaattcca gctccaatag tgtatattaa 601 agttgctgtg attaaaacgc tcgtagttga attaaaatgt gattttatac attttgaaga 661 ctttanntaa gtgaagtttc tagaaatgtt aaattaaaat caaagaagga gacnnttcaa 721 gtaattgagt tgttattact ttgaataaaa taaggtgttt aaagcaaaac attatgttaa 781 tgaatattcg agcatgggac aatgctgagg ggatgtcaat tagacatttc gagagaagga 841 ttaaaaggaa caattggggt gattcagaaa ataacgggag aggtgaaaat ccatgatcgg 901 tataagatgc acgagagcga aagcatttca ctcaactggg tccattaatc aagaacgaaa 961 gttaggggat cgaagacgat cagataccgt cgtagtccta actataaacg atgtcaacca 1021 aggattggat gaaattcaga tgtacaaaga tgaagaaaca ttgtttctaa atccaagtat 1081 atcaatacta ccttgttcag aacttaaaga gaaatcttga gtttatggac ttcaggggga 1141 gtatggtcac aaggctgaaa cttaaaggaa ttgacggaag ggcacaccag gagtggagcc 1201 tgcggcttaa tttgactcaa cacgggaaaa cttaccaaga ccgaacagta gaaggaatga 1261 cagattaaga gttctttcat gatttattgg gtagtggtgc atggccgttc ttagttggtg 1321 gagtgatttg tcaggttaat tccggtaacg aacgagactg aaacctatta attagttttc 1381 tgcctataag acagaaatgt tcgcaagaac aggtgcgtaa gtaccacttc ttaaagggac 1441 acatttcaat tgtcctattt taattgttag ttatctaatt tcgattagaa ctcttttaac 1501 gtgggaaaaa gaaaaaggaa gcattcagca ataacaggtc tgtgatgccc ttagacatct 1561 tgggccgcac gcgcgctaca atggagttac tagagagcat tttatcattt acaccttatt 1621 tattaggcta tgtctaatag gtagggatag taagtggtgt accgagattg aaatagttaa 1681 ggaaaactca aaagaacgta catgacaggg ataaatgatt ggaattattt gttttgaacg 1741 aggaattcct tgtaatatcg agtcattaac tcgagatgaa tacgtccctg ccctttgtac 1801 acaccgcccg tcgctcctac cgattgaata aagaggtgaa attctaggat tctgtcttat 1861 agatagaaaa atggatttaa atctccttat ttagaggaag gagaagtcgt aacaaggttt 1921 ccgtaggtga acctgcggaa ggatcatta

Get the E. histolytica rRNA, DNA sequence X56991

X56991. Reports E.histolytica gen...[gi:9283] Links

LOCUS EH16SRRNA 1947 bp DNA linear INV 29-OCT-1992DEFINITION E.histolytica gene for small subunit ribosomal RNA (16S-like).ACCESSION X56991VERSION X56991.1 GI:9283KEYWORDS 16S ribosomal RNA homologue; ribosomal RNA; small subunit ribosomal RNA.SOURCE Entamoeba histolytica ORGANISM Entamoeba histolytica Eukaryota; Entamoebidae; Entamoeba.REFERENCE 1 AUTHORS Sogin,M.L., Edman,U., Elwood,H.E. and Agabian,N. TITLE Small subunit ribosomal RNA from Entamoeba histolytica JOURNAL Nucleic Acids Res.REFERENCE 2 (bases 1 to 1947) AUTHORS Sogin,M.L. TITLE Direct Submission JOURNAL Submitted (14-DEC-1990) M.L. Sogin, MARINE BIOLOGICAL LAB, CENTER FOR MOL EVOLUTION, WOODS HOLE MA 02543, U S AFEATURES Location/Qualifiers source 1..1947 /organism="Entamoeba histolytica" /mol_type="genomic DNA" /db_xref="taxon:5759" rRNA 1..1947 /product="small subunit ribosomal RNA (16S-like)

ORIGIN 1 tatctggttg atcctgccag tattatatgc tgatgttaaa gattaagcca tgcatgtgta 61 agtataaaga ccaagtagga tgaaactgcg gacggctcat tataacagta atagtttctt 121 tggttagtaa aatacaagga tagctttgtg aatgataaag ataatacttg agacgatcca 181 gtttgtatta gtacaaaatg gccaattcat tcaatgaatt gagaaatgac attctaagtg 241 agttaggatg ccacgacaat tgtagaacac acagtgttta acaagtaacc aatgagaatt 301 tctgatctat caatcagttg gtagtatcga ggactaccaa gattataacg gataacgagg 361 aattggggtt cgacatcgga gagggagctt tacagatggc taccacttct aaggaaggca 421 gcaggcgcgt aaattaccca ctttcgaatt gaagaggtag tgacgacaca taactctaga 481 gttgagtaaa atcaattctt gaaggaatga gtaggaggta aattctccta cgaaatcaat 541 tggagggcaa gtctggtgcc agcagccgcg gtaattccag ctccaatagt gtatattaaa 601 gttgctgtga ttaaaacgct cgtagttgaa ttaaaatgtg gttttataca ttttgaagac 661 tttatgtaag taaagtttct agaaatgtta aattaaaatc aaagaaggaa acaattcaag 721 taattgagtt gttattactt tgaataaaat aaggtgttta aagcaaaaca ttatgttaat 781 gaatattcaa gcatgggaca atgctgaggg gatgtcaata agacatttcg agagaaggat 841 taaaaggaac aattggggtg attcagaaaa taacgggaga ggtgaaaatc catgatcgct 901 ataagatgca cgagagcgaa agcatttcac tcaactgtgt ccattaatca agaacgaaag 961 ttaggggatc gaagacgatc agataccgtc gtagtcctaa ctataaacga tgtcaaccaa 1021 ggattggatg aaattcagat gtacaaagat agagaagcat tgtttctaga tctgagtata 1081 tcaatattac cttgttcaga acttaaagag aaatcttgag tttatggact tcagggggag 1141 tatggtcaca aggctgaaac ttaaaggaat tgacggaagg gcacaccagg agtggagcct 1201 gcggcttaat ttgactcaac acgggaaaac ttaccaagac cgaacagtag aaggaatgac 1261 agattaagag ttctttcatg atttattggg tagtggtgca tggccgttct tagttggtgg 1321 agtgatttgt caggttaatt ccggtaacga acgagactga aacctattaa ttagttttct 1381 gcctataaga cagaaatgtt cgcaagaaca ggtgcgtaag taccacttct taaagggaca 1441 catttcaatt gtcctatttt aattgtagtt atctaatttc ggttagacct cttttaacgt 1501 gggaaaaaga aaaaggaagc attcagcaat aacaggtctg tgatgccctt agacatcttg 1561 ggccgcacgc gcgctacaat ggagttacta gagagtattt tatcatttac accttattta 1621 ttaggctttg tctaataatt aaggatagta agtggtgtac cgagattgaa atagttaagg 1681 aaaactcaaa agaacgtaca tgacagggat aaatgattgg aattatttgt tttgaacgag 1741 gaattccttg taatatcgag tcattaactc gagatgaata cgtccctgcc ctttgtacac 1801 accgcccgtc gctcctaccg attgaataaa gaggtgaaat tctaggattc tgtcttatag 1861 atagaaaaat ggatttaaat ctccttattt agaggaagga gaagtcgtaa caaggtttcc 1921 gtaggtgaac ctgcggaagg atcatta//

blastThe Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences.The program compares nucleotide or protein sequences to sequence databases and calculatesthe statistical significance of matches.BLAST can be used to infer functional and evolutionary relationships between sequencesas well as help identify members of gene families.

Query: 1 tatctggttgatcctgccagtattatatgctgatgttagagattaagccatgcatgtgta 60 |||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||Sbjct: 1 tatctggttgatcctgccagtattatatgctgatgttaaagattaagccatgcatgtgta 60

Query: 61 agtataaagaccaagtaggatgaaactgcggacggctcattataacagtaatagtttctt 120 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 61 agtataaagaccaagtaggatgaaactgcggacggctcattataacagtaatagtttctt 120

Query: 121 tggttagtaaagtacaaggatagctttgtgaatgataaagataatacttgagacgatcca 180 ||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 121 tggttagtaaaatacaaggatagctttgtgaatgataaagataatacttgagacgatcca 180

Query: 181 atttgtattagtacaaagtggccaatttatgtaagtaaattgagaaatgacattctaagt 240 |||||||||||||||| ||||||||| || || | |||||||||||||||||||||||Sbjct: 181 gtttgtattagtacaaaatggccaattcattcaa-tgaattgagaaatgacattctaagt 239

Query: 241 gagttaggatgccacgacaattgtagaacacacagtgtttaacaagtaaccaatgagaat 300 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 240 gagttaggatgccacgacaattgtagaacacacagtgtttaacaagtaaccaatgagaat 299

Query: 961 gttaggggatcgaagacgatcagataccgtcgtagtcctaactataaacgatgtcaacca 1020 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 960 gttaggggatcgaagacgatcagataccgtcgtagtcctaactataaacgatgtcaacca 1019

Query: 1021 aggattggatgaaattcagatgtacaaagatgaagaaacattgtttctaaatccaagtat 1080 ||||||||||||||||||||||||||||||| |||| ||||||||||| ||| |||||Sbjct: 1020 aggattggatgaaattcagatgtacaaagatagagaagcattgtttctagatctgagtat 1079

Query: 1081 atcaatactaccttgttcagaacttaaagagaaatcttgagtttatggacttcaggggga 1140 ||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 1080 atcaatattaccttgttcagaacttaaagagaaatcttgagtttatggacttcaggggga 1139

Blg I site agatct (agatct is a palindrome) is only present in Entamoeba histolyticaDNA strider data.

1. What are the differences between DNA pol and RT-DNA pol ?2. Cites the activities of E. Coli DNA polymerase I 3. What is the rate of DNA synthesis of the E. Coli DNA polymerase I.4. What does “proofreading activity” means”?5. What do “nick translation and Taqman activity” mean”?6. Why is Pfu DNA pol 10 times less active than Taq DNA pol ?7. What’ s a palindromic element ?8. What‘s a SNP within a palindrome.9. Explain the reaction catalyzed by T4 DNA ligase and ATP.10. Why is it better for a mammalian vector to have an intron before the insertion site ?11.explain The „cut and paste“ reaction in molecular biology.12. Why are there sometimes T4 and SP6 promoter elements at both ends of an insertion site ?13. What does “DNA dependant” means in “DNA dependant RNA pol” ?14. Does the RNA pol need a primer ?15. How does topoisomerase I and II work ?

questions

main enzymes used in molecular biology (theory and practice) by jean-pierre herveg and a lot of...

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