1. Enzymes for Genetic Engineering
Chapter 2
Enzymes for Genetic Engineering
2.1 Restriction endonucleases
2.2 DNA ligase
2.3 DNA polymerase
2.4 Other Enzymes
2.5 probe labeling techniques

2. 第二章 基因工程的工具酶
教学目的、要求：
1. 了解基因工程各种工具酶的基本概念、分类及在基因工程中的作用；
2．掌握限制性内切酶，聚合酶，连接酶，反转录酶，修饰酶的生物学特性和应用；
教学内容：
1、工具酶与基因工程
2、限制酶
3、DNA聚合酶
4、DNA连接酶
5、S1核酸酶
6、BAL31核酸酶
7、碱性磷酸酶
8、逆转录酶
9、T7和SP6RNA聚合酶

3. Enzymes for DNA analysis and molecular cloning
function
Application
Restriction endonucleases
fragment DNA into defined segments
cloning
DNA Ligase
joins DNA fragments
DNA polymerase
RNA polymerase
synthesizes DNA based on a template
PCR
fills in gaps in DNA
Reverse transcriptase
copies RNA into DNA
􀃆 cDNA; RT-PCR
Terminal transferase
add poly nucleotides
for radiolabeling
Polynucleotide kinase
transfer P from ATP to the 5' end
For radiolabeling
Alkaline phosphatase
remove phosphate
Avoid self ligation
S1 nuclease
Cut single-stranded DNA and RNA
Ribonucleases (endo and exo RNase) 􀃆
degradation of RNA into smaller components

4. 2.1 Restriction endonucleases
2.1.1 Restriction modification system (R-M )
2.1.2 nomenclature
2.1.3 three types of RE
2.1.4 Factors Affecting Restriction Enzyme
Digestion

5. 2.1 Restriction endonucleases
(限制性核酸内切酶）
Restriction Enzymes are:
- Proteins
- Cleave DNA inside with a sequence-specific manner
- Different restriction enzymes in different organisms
- Evolved as a defense mechanism against infection by foreign viruses
- Isolated from bacteria，algae and archaea (古生菌 )
Extreme bacteria
since the Archaea have an independent evolutionary history and show many differences in their biochemistry from other forms of life, they are now classified as a separate domain in the three-domain system. In this system the phylogenetically distinct branches of evolutionary descent are the Archaea, Bacteria and Eukarya
After isolating the first restriction enzyme, HindII, in 1970, and the subsequent discovery and characterization of numerous restriction endonucleases, led to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria.

6. Facts:
the first restriction enzyme, HindII, was isolated in 1970 by Hamilton Smith and his colleagues [1]
the 1978 Nobel Prize for Physiology or Medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton Smith.
Over 3000 restriction enzymes have been studied in detail
More than 600 of these are available commercially
Routinely used for DNA modification and manipulation in
laboratories.
Roberts RJ (April 2005). "How restriction enzymes became the workhorses of molecular biology". Proc. Natl. Acad. Sci. U.S.A. 102 (17): 5905–8.
Roberts RJ, Vincze T, Posfai J, Macelis D. (2007). "REBASE--enzymes and genes for DNA restriction and modification". Nucleic Acids Res 35 (Database issue): D269–70.

7. 2.1.1 Restriction modification system (限制与修饰系统）
Restriction Enzymes have evolved to provide a defense mechanism against invading viruses. [1]
Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction.
Host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity.
Collectively, these two processes form the restriction modification system (R-M system).
Arber W, Linn S (1969). "DNA modification and restriction". Annu. Rev. Biochem. 38: 467–500.
Kobayashi I (September 2001). "Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution". Nucleic Acids Res. 29 (18): 3742–56

8. Bacteria phage‘s invation causes host cells dead and lead to form plaques.
Lawn 菌膜
Plaque 嗜菌斑

9. W. Arber and S. Linn (1969) E. coli C
Plating efficiencies of bacteriophage λ (l phage) grown on E. coli strains C, K-12 and B:
Phageλ E.Coli C plaque （＋）
E.Coli K plaque （－）
E.Coli B plaque （－）
E.coliK-12 and B can resist phage λ.
The DNA of phage which had been grown on strains K-12 and B were found to have chemically modified bases which were methylated.
Working with one of his graduate students, Daisy Dussoix, Arber found in 1962 that restriction was host-controlled and involved changes in the phage's DNA. In effect, the DNA of the invading phages is cut into component parts, although some phages survived the operation. This discovery set in motion a series of studies that jump-started genetics to become the new frontier in biomedical research. Read more: Werner Arber Biography (1929-)

10. Additional studies with other strains indicate that different strains had specific methylated bases.
EcoRI
vs.
EcoRI Methylase
A RE specificlly paired with a methylase. This mechanism prevents the REs from cleaving up the host DNA.
R-M for this strain
5’—GAAmTTC—3’ will not be cut by EcoRI
3’—CTTAmAG—5’

11. A characteristic feature of the sites of methylation, was that they involved palindromic（回文） DNA sequences. EcoR1 methylase specificity（Rubin and Modrich, 1977）
The REs cleaved at or near the methylation recognition site. However, they would not cleave at these specific palindromic sequences if the DNA was methylated.
In addition to possessing a particular methylase, individual bacterial strains also contained accompanying specific endonuclease activities.

12. Methylation occurres at very specific sites in the DNA
Typical sites of methylation include the N6 position of adenine, the N4 position, or the C5 position of cytosine.
Assignment1: what are the methylating sites for Guanine and Thymine?

13. 2.1.2 Nomenclature (命名）
Since their discovery in the 1970s, many different restriction enzymes have been identified in different bacteria. Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus（属）, species（种） and strain（菌株）.
eg. EcoRI restriction enzyme was derived as follow
Abbreviation
Meaning
Description E
Escherichia
genus co
coli
species R
RY13
strain I
First identified
order of identification in the bacterium

14. Each restriction enzyme has specific recognition sequence
Enzymes with 6-bp Recognition sequence
Bgl II “bagel-two” Bacillus globigi ’-AGATCT-3’
3’-TCTAGA-5’
Pst I “ P-S-T-one” Providencia stuartii ’-CTGCAG-3’
3’-GACGTC-5’
Enzyme with4-bp Recognition sequence
Hha I “ha-ha-one” Haemphilus haemolyticus ’-GCGC-3’
3’-CGCG-5’
Sau3A “sow-three-A” Staphylococcus aureus 3A ’-GATC-3’
3’-CTAG-5’
Enzyme with 8-bp Recognition sequence
Not I “not-one” Nocardia ’-GCGGCCGC-3’
otitidis-caviarum ’-CGCCGGCG-5’

15. 2.1.3 Types of Restriction Enzymes
Restriction endonucleases are categorized into three general groups (Types I, II and III) based on
composition (protein subunits)
enzyme cofactor requirements (Mg2+, SAM, ATP)
the nature of their target sequence
the position of their DNA cleavage site relative to thetarget sequence.

16. Type I restriction enzymes
Possess three subunits called HsdR, HsdM, and HsdS:
HsdR is required for restriction
HsdM is necessary for adding methyl groups to host DNA
HsdS is important for specificity of cut site recognition in
addition to its methyl transferase activity.
multifunctional enzymes, multimers
Enzyme cofactors required for their activity:
S- Adenosyl methionine (AdoMet or SAM)
hydrolyzed adenosine triphosphate (ATP)
magnesium ions (Mg2+ )
Me doner
Energy doner
Cleavage activity activator

17. Target sequence:
The recognition site is asymmetrical and is composed of two portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a spacer of about 6–8 nucleotides.
eg. EcoB:
5’-TGANNNNNNNNTGCT-3’
Cleavage site:
cut at a site that differs, and is some distance (at least 1000 bp) away, from their recognition site.
Recognition site ≠ Cutting site

18. Type III restriction enzymes
Contain more than one subunit
Double functions (cut and methylase)
Require Mg2+, AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively
Recognize two separate non-palindromic sequences that are inversely oriented （反向排列）.
e.g. EcoP1: AGACC
EcoP15 : CAGCAG
Cut DNA about base pairs out side the recognition site with unpredictable manner.

19. Type II restriction enzymes
Dimers (consists of two identical subunits)
Usually require only Mg2+ as a cofactor for their
enzyme activity
Restriction activity but no modification
Recognition sites are usually undivided and
palindromic and 4–8 nucleotides in length
Each cuts in a predictable manner, at a site
within or adjacent to the recognition sequence
The most commonly available and used restriction enzymes
The classic II .

20. Structure of EcoRI dimer

21. In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes. These subgroups are defined using a letter suffix.
Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than one subunit. They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg2+ cofactors. Type IIE restriction endonucleases (e.g. NaeI) cleave DNA following interaction with two copies of their recognition sequence. One recognition site acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or improves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both sequences at the same time.Type IIG restriction endonucleases (Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active. Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA.Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites. These enzymes may function as dimers. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits. Some recognize palindromic sequences while others have asymmetric recognition sites

22. Commonly used RE, Recognition Sites

23. Occurring frequencies of recognition sites
Recg sites
Sequence
Frequency A
A,G,C,T
1/4 AT
A-A/G/C/T; G-A,G,C,T
C-A/G/C/T; T-A,G,C,T
1/42
ATT
1/43
AATT
1/44
AAGTT
1/45 … nN
1/4n
E.Coli: genome DNA 4.2 x 106 bp, EcoRI frequency:
4.2 x 106 x 1/46 = 4.2 (cut sites in whole genome DNA)

24. Examples Eco RI Hae III 2 cuts with 3 fragments.
GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA
CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT
Eco RI
GGCCTGCG AATTCCCGATCGAAGGCCCG AATTCTGGCCA
CCGGACGCTTAA GGGCTAGCTTCCGGGCTTAA GACCGGT
2 cuts with 3 fragments.
GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA
CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT
Hae III
GG CCTGCGAATTCCCGATCGAAGG CCCGAATTCTGG CCA
CC GGACGCTTAAGGGCTAGCTTCC GGGCTTAAGACC GGT
3 cuts with 4 fragments.

25. Two major ways for RE cleave
“blunt ends”
Symmetrical cutting

26. “Sticky ends”-I
Asymmetrical cutting
3’OH of the sticky end is very important for re-joining of DNA fragments.
5’-P: required for phosphodiester bond formation
5’- overhanging sticky ends

27. “Sticky ends”-II
Pst1
Providencia stuartii
3’overhanging sticky end

28. 2.1.4 Factors Affecting Restriction Enzyme Digestion
DNA Purity
Cross contamination
Metylation: dam(+) dcm(+) strains
GATC  GAmTC; CCWGG  CCmWGG
BamHI  GGATCC ?
Temperature and time
Buffer: will give the optimum pH, ionic strength, Mg2+,
Star activities: a relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimum for the enzyme.
Reaction volume and RE amount
W=Purine base

29. 2.2 DNA ligase
DNA ligase is a special type of ligase, which is basically an enzyme that in the cell repairs single-stranded discontinuities in double stranded DNA molecules ( strands that have double-strand break, a break in both complementary strands of DNA).
Purified DNA ligase is used in gene cloning to join DNA molecules together. The alternative, a single-strand break, is fixed by a different type of DNA ligase using the complementary strand as a template but still requires DNA ligase to create the final phosphodiester bond to fully repair the DNA.
Okazaki fragment （冈崎片段？）

30. Ligase mechanism nick: lack P-diester bound gap: nucleotide missing 缺刻3’ 5’ 5’ 5’ 5’ 3’
nick: lack P-diester bound
gap: nucleotide missing 缺刻 缺口
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another.

31. Ligation needs:
Energy-dependent joining of the chains
Activated by NAD+ or ATP hydrolysis
E. coli DNA lygase: NAD  NMN+ + AMP
T4 DNA lygase: ATP  AMP + PPi
AMP -attaches to lysine group on enzyme
AMP transferred to 5’ phosphate at ligation site
3’ OH at ligation site splits out AMP and joins to 5’ phosphate

32. Activated Phosphorylating complex AMP-Lys-ligase complex
Ligase Mechanism +
NH2-Lysine-
High Energy
Nitrogen Phosphate
Bond
NAD+
NMN+ O - P C H 2 N
NH2-Lysine-
Activated Phosphorylating
complex
AMP-Lys-ligase complex

33. P O OH O - P C H 2 N
NH2-Lysine-

34. P O OH - C H 2 N
NH2-Lysine- O P
+ AMP

35. The formation of phosphodiester bound
ATP is required for the ligase reaction.

36. In general, 14-16 °C, over night (o/n)
One vital, and often tricky, aspect to performing successful recombination experiments involving the ligation of cohesive-ended fragments is controlling the optimal temperature.
Most experiments use T4 DNA Ligase (isolated from bacteriophage T4), which is most active at 25°C. However, in order to perform successful ligations with cohesive-ended fragments, the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the DNA fragments being ligated.
In general, °C, over night (o/n)
Thinking: Why the temperature is the critical factor affecting ligation?

37. If the ambient（周围）temperature exceeds Tm, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding.
Since blunt-ended DNA fragments have no cohesive ends to anneal, controlling the optimal temperature becomes much less important. The most efficient ligation temperature will be the temperature at which T4 DNA ligase functions optimally. Therefore, the majority of blunt-ended ligations are carried out at 20-25°C.

38. Joining of stick ends and blunt ends
Use of linkers:

39. Use of adaptors: when the restriction enzyme can also cut the DNA fragment, using an adaptor to join DNA fragments may considered.
an adaptor containing sticky end with 5’-OH modified terminus to avoid self ligation.

40. Produce sticky ends by homopolyer tailling:
use of terminal deoxylnucleotidyl transferase
Add a series of poly nucleotides onto the 3’-terminal of a dsDNA.
Only need to add one dNTP into the test tube when conduct the polymerase catalyzing reaction.

41. Topoisomerase mediated TA cloning:
Topoisomerase is both a restriction enzyme and ligase naturally involving in DNA replication.
Topoisomerase I from vaccinia virus binds dsDNA at specific sequence and cleave it after the 5’-CCCTT in one strand.
The energy from the broken bond is conserved by formation of a covalent bond between the 3’- phosphate of the cleavaged DNA strand and the 274 tyrosine residue of topoisomerase I.
The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5’ hydroxyl of the original cleaved strand , reversing the reaction and releasing topoisomerase. (Shuman, )

42. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single A to the 3’-ends of PCR products.
Linearized vector DNA with overhanging 3’-T, and covalently bound to the topoisomerase I
PCR products inserts to ligate efficiently with the vector.
RT: 5 min,
Vol: 6 ul

43. PCR products inserts to ligate efficiently with the vector.

44. topoisomerase, originally termed gyrase, was first discovered by Taiwanese Harvard Professor James C. Wang.[
The double-helical configuration that DNA strands naturally reside in makes them difficult to separate, and yet they must be separated by helicase proteins if other enzymes are to transcribe the sequences that encode proteins, or if chromosomes are to be replicated. In so-called circular DNA, in which double helical DNA is bent around and joined in a circle, the two strands are topologically linked, or knotted. Otherwise, identical loops of DNA having different numbers of twists are topoisomers, and cannot be interconverted by any process that does not involve the breaking of DNA strands. Topoisomerases catalyze and guide the unknotting of DNA by creating transient breaks in the DNA using a conserved Tyrosine as the catalytic residue.
Type I topoisomerase cuts one strand of a DNA double helix, relaxation occurs, and then the cut strand is reannealed.
Type II topoisomerase cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then reanneals the cut strand. It is also split into two subclasses: type IIA and type IIB topoisomerases, which share similar structure and mechanisms. Examples of type IIA topoisomerases include eukaryotic topo II, E. coli gyrase, and E. coli topo IV. Examples of type IIB topoisomerase include topo VI.

45. 2.3 DNA polymerase
DNA polymerase: an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand alone the template DNA strand.
DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand. The newly-polymerized molecule is complementary to the template strand and identical to the template's original partner strand.
DNA polymerases use a magnesium ion for catalytic activity.

46. 2.3.1 DNA polymerase I and Klenow fragment
Structure:
single peptide with different domains
Functions:
5’3' Polymerase activity
3’5' exonuclease (proofreading)
5’3' exonuclease activity (RNA Primer remove)
DNA polymerase can add a nucleotide onto only a preexisting 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide.

47. Reaction condition for noncell system:
4 dNTPs (substrates)
Mg2+
Peimers with 3’-OH
(no known DNA polimerase is able to begin a new chain)
DNA template: template reading 3’ 5’
new chain extension 5’3’

48. The Klenow fragment is a large protein fragment produced when DNA pol I from E. coli is enzymatically cleaved by the protease subtilisin.

49. Because the 5' → 3' exonuclease activity of DNA pol I makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research.
The Klenow fragment is extremely useful for research-based tasks such as:
Synthesis of double-stranded DNA from ssDNA
templates
Filling in (meaning removal of overhangs to create
blunt ends) recessed 3' ends of DNA fragments
Digesting away protruding（凸出的） 3' overhangs
Preparation of radioactive DNA probes

50. 2.3.2 T4 and T7 DNA polymerase Source: Bacterophageies of E.coli
Template dependent ＤNA　polymerase
T4 DNA polymerase
Function：Similar to Klenow fagment 　
3’-5’ cleavage (exonuclease)
Characteristics：In general, T4 DNA polymerase is used for the same types of reactions as Klenow fragment, particularly in blunting the ends of DNA with 5' or 3' overhangs.

51. two differences between the two enzymes that have practical signficance:
The 3' -> 5' exonuclease activity of T4 DNA polymerase is roughly 200 times that of Klenow fragment, making it preferred by many investigators for blunting DNAs with 3' overhangs.
While Klenow fragment will displace（shift） downstream oligonucleotides as it polymerizes, T4 DNA polymerase will not. This attribute makes T4 DNA polymerase the more efficient choice for certain types of oligonucleotide mutagenesis reactions.

52. T7 DNA polymerase
a DNA-dependent DNA polymerase responsible for the fast rate of T7 phage DNA replication in vivo.
the 3' -> 5' are approximately 1000-fold greater than that of Klenow fragment.
The polymerase consists of a 1:1 complex of the viral T7 gene 5 protein (80kDA) and the E. coli thioredoxin (12kDA).
high fidelity and strand displacement synthesis prevention

53. This polymerase is unique due to its considerable processivity, or ability to stay on DNA for a greater than average number of base pairs. This makes it particularly useful for recombinant protein expression systems
Using the T7 promoter and T7 polymerase strongly drive the inserted gene of interest without inducing host protein overexpression.
It is also suitable for site-directed mutagenesis.

54. Taq DNA polymerase
a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965.
Taq's optimum temperature for activity is ℃, with a half life of 9 minutes at 97.5 ℃ , and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72 ℃
One of Taq‘s drawbacks（弊端） is its relatively low replication fidelity. It lacks a 3'- 5‘ exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides

55. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity（高保真） amplification.
Taq makes DNA products that have A (adenine) overhangs at their 3' ends. This may be useful in TA cloning
Pfu makes blunt PCR fragments
Put these two enzymes together ？

56. 2.3.4 Reverse Transcriptase
A reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into complimentary DNA (cDNA). It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA.
Reverse transcriptase was discovered by Howard Temin at the University of Wisconsin–Madison, and independently by David Baltimore in 1970 at MIT. The two shared the 1975 Nobel Prize in Physiology or Medicine with Renato Dulbecco for their discovery.

57. Functions: RNA-dependent DNA ploymerase;
RNase H activity: removing RNA from the RNA-DNA hybrids.
DNA polymerase activity

58. RNA retroviruses
eg., HIV, breast cancer
The process of reverse transcription is extremely error-prone (易于出错) and it is during this step that mutations may occur.
Host cell can use its DNA polymerase to synthesis the second strand DNA and displace the RNA from the cDNA.

59. Well studied transcriptases:
HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV)
M-MLV reverse transcriptase from the Moloney murine leukemia virus (鼠白血病病毒)
AMV reverse transcriptase from the avian myeloblastosis virus (禽成髓细胞瘤病毒)
Telomerase reverse transcriptase that maintains the telomeres of eukaryotic chromosomes
The highlighted two are well commercialized and routinely used in molecular cloning.

60. Applications Target of antivirus drugs
As HIV uses reverse transcriptase to copy its genetic material and generate new viruses (part of a retrovirus proliferation circle), specific drugs have been designed to disrupt the process and thereby suppress its growth. Collectively, these drugs are known as reverse transcriptase inhibitors and include the nucleoside and nucleotide analogues(类似物) zidovudine (trade name Retrovir), lamivudine (Epivir) and tenofovir (Viread), as well as non-nucleoside inhibitors, such as nevirapine (Viramune).

61. Reverse transcriptase is commonly used in research to apply the polymerase chain reaction technique to RNA in a technique called reverse transcription polymerase chain reaction (RT-PCR) as well as real-time PCR.
Reverse transcriptase is used also to create cDNA libraries from mRNA. The commercial availability of reverse transcriptase greatly improved knowledge in the area of molecular biology, as, along with other enzymes, it allowed scientists to clone, sequence, and characterize DNA.

62. 2.4 Other Enzymes 2.4.1 Alkaline phosphatase (ALP, ALKP):
A hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides (DNA, RNA), proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation.
Alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase.
Ribbon diagram (N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase

63. BAP: isolated from bacteria
BAP: isolated from bacteria. In bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell membrane. BAP is comparatively resistant to inactivation, denaturation, and degradation, and also has a higher rate of activity.
The optimal pH for the activity of the E. coli enzyme is 8.0
CIP: derived from Calf Intestinal Alkaline Phosphatase. the bovine enzyme optimum pH is slightly higher at 8.5

64. P O OH - C H 2 N O P OH
+ AMP

65. EcoRI HO
5’-pAATTC P P OH
GAATTCCTTAAG HO P
EcoRI OH OH 5’ P P 5’ OH OH OH P
5’-pAATTC

66. Use in research
A useful tool to remove 5’- phosphates and prevent the DNA from ligating, thereby keeping DNA molecules linear until the next step of the process for which they are being prepared
Radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.
Diagnostic uses:

67. 2.4.2 S1 Nuclease
An endonuclease that is active against single-stranded DNA and RNA molecules. It is five times more active on DNA than RNA.
The reaction products are oligonucleotides or single nucleotides with 5' phosphoryl groups.
It can also occasionally introduce single-stranded breaks in double-stranded DNA or RNA, or DNA-RNA hybrids.
It is used as a reagent in nuclease protection assays, removing single stranded tails from DNA molecules to create blunt ends and opening hairpin loops.

68. 2.4.3 DNase and RNase
DNase: An endonuclease that is active against dsDNA and ssDNA molecules.
Heat inactive; EDTA inhibition
The reaction products: oligonucleotides or single nucleotides with 5' phosphoryl groups.
Use DNase free stuffs to perform the DNA cloning.
DNase I is commonly used in DNA foot-printing and in RNA extraction

69. DNase I in DNA foot-printing

70. DNase I in DNA foot-printing
An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract.
The unprotected DNA is then digested with DNase I such that on average, every DNA molecule is cut once.
Digestion products are then resolved by electrophoresis.
Comparison of the DNase I digestion pattern in the presence or absence of protein will allow the identification of a footprint (protected region).

71. RNase (Ribonuclease): A type of nuclease that catalyzes the degradation of RNA into smaller components.
endoribonucleases
RNases
exoribonucleases,
comprise several sub-classes of the phosphorolytic enzymes and of the hydrolytic enzymes.
one of the hardiest enzymes in common laboratory usage : heat resistant; extremely common;
In addition, active RNA degradation systems are a first defense against RNA viruses, and provide the underlying machinery for more advanced cellular immune strategies such as RNAi.
RNA degradation is a very ancient and important process. As well as cleaning of cellular RNA that is no longer required, RNases play key roles in the maturation of all RNA molecules, both messenger RNAs, and non-coding RNAs that function in varied cellular processes.

72. RNases are extremely common, resulting in very short lifespans for any RNA that is not in a protected environment.
It is worth noting that all intracellular RNAs are protected from RNase activity by a number of strategies including 5' end capping, 3' end polyadenylation, and folding within an RNA protein complex (ribonucleoprotein particle or RNP).

73. RNase A an RNase that is commonly used in research.
It is sequence specific for single-stranded RNAs. It cleaves 3'end of unpaired C and U residues, leaving a 3'-phosphorylated product.
RNase H a ribonuclease that cleaves the RNA in a DNA/RNA duplex to produce ssDNA.
RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism, aided by an enzyme-bound divalent metal ion. RNase H leaves a 5'-phosphorylated product.
RNase I cleaves 3'-end of ssRNA at all dinucleotide bonds leaving a 5' hydroxyl, and 3' phosphate.

74. 2.5 Nucleic probes
Nucleic probe: In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length (usually bases long), which is used to detect the presence of the DNA target nucleotide sequences that are complementary to the sequence in the probe.
The probe hybridizes to ssDNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target.
The labeled probe is first denatured (by heating or under alkaline conditions) into single DNA strands and then hybridized to the target DNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ.

75. To detect hybridization of the probe to its target sequence, the probe is tagged (or labelled) with a molecular marker of either radioactive or (more recently) fluorescent molecules
commonly used markers are 32P or Digoxigenin, which is non-radioactive antibody-based marker.
DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via autoradiography or other imaging techniques.

76. 硝酸纤维膜

78. Probes labeling techniques
Depending on the method the probe may be synthesized using phosphoramidite method or generated and labeled by PCR amplification. Molecular DNA- or RNA-based probes are now routinely used in screening gene libraries, detecting nucleotide sequences with blotting methods, and in other gene technologies like microarrays.

79. Nick Translation： carefully using DNase I digest DNA to produce nicks, then, using the DNA polymerase and dNTP (one of which was radio-labeled). The nick may shift as the enzyme catalyzing deoxylnucleotidyl on the 3’-end.

80. End Filling: a gentaler method than nick translation and rarely causes breakage of the DNA, but unfortunately can only be use to label DNA molecules that have sticky ends.

81. Randome priming: using random synthesized hexameric oligonucleotides (6 聚寡核苷酸）as primers to anneal with an DNA fragments, run PCR by klenow fragment to produce radiolabeling probes. (to get a probe pool)

82. 抗生物素蛋白