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
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
Enzymes for DNA analysis and molecular cloning
Enzyme
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
Reverse transcriptase
copies RNA into DNA
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
PCR
fills in gaps in DNA
cDNA; RT-PCR
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
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 )
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.
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
Bacteria phage‘s invation causes host cells dead and lead to form plaques
Lawn
菌膜
Plaque 嗜菌斑
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
E.Coli K-12
E.Coli B
plaque （＋）
plaque （－）
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.
• Additional studies with other strains indicate that
different strains had specific methylated bases.
EcoRI
vs.
EcoRI Methylase
R-M for this strain
5’—GAAmTTC—3’ will not be cut by EcoRI
3’—CTTAmAG—5’
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.
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?
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
E
Escherichia
Description
genus
co
coli
species
R
RY13
strain
I
First identified
order of identification
in the bacterium
Each restriction enzyme has specific recognition sequence
Enzymes with 6-bp Recognition sequence
Bgl II
“bagel-two”
Bacillus globigi
5’-AGATCT-3’
3’-TCTAGA-5’
Pst I
“ P-S-T-one”
Providencia stuartii
5’-CTGCAG-3’
3’-GACGTC-5’
Enzyme with4-bp Recognition sequence
Hha I
“ha-ha-one”
Haemphilus haemolyticus
Sau3A “sow-three-A” Staphylococcus aureus 3A
Enzyme with 8-bp Recognition sequence
Not I
“not-one”
Nocardia
otitidis-caviarum
5’-GCGC-3’
3’-CGCG-5’
5’-GATC-3’
3’-CTAG-5’
5’-GCGGCCGC-3’
3’-CGCCGGCG-5’
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.
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
Target sequence:
Recognition site ≠ Cutting site
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.
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 20-30 base pairs out side the recognition
site with unpredictable manner.
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
Structure of EcoRI dimer
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
Commonly used RE, Recognition Sites
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)
Examples
Eco RI
GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA
CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT
GGCCTGCG
AATTCCCGATCGAAGGCCCG
CCGGACGCTTAA
GGGCTAGCTTCCGGGCTTAA
AATTCTGGCCA
GACCGGT
2 cuts with 3 fragments.
Hae III
GG
CC
GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA
CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT
CCTGCGAATTCCCGATCGAAGG
GGACGCTTAAGGGCTAGCTTCC
3 cuts with 4 fragments.
CCCGAATTCTGG
GGGCTTAAGACC
CCA
GGT
Two major ways for RE cleave
 “blunt ends”
Symmetrical cutting
“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
“Sticky ends”-II
Pst1
Providencia stuartii
3’overhanging sticky end
2.1.4 Factors Affecting Restriction Enzyme Digestion
 DNA Purity
 Cross contamination
 Metylation: dam(+)
dcm(+) strains
GATC  GAmTC; CCWGG  CCmWGG
BamHI  GGATCC ?
W=Purine base
 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
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？）
Ligase mechanism
3’
5’
5’
5’
3’
nick: lack P-diester bound
缺刻
5’
gap: nucleotide missing
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.
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
Ligase Mechanism
NH2
N
N
N
CONH2
O
N
NH2-Lysine-
O
O-P-O-P-O CH2 N +
O CH2
O
O O
HO
OH
High Energy
Nitrogen Phosphate
Bond
HO OH
NAD+
NH2
NMN+
N
N
N
O
N
O-PO CH2
O
HO
NH2-Lysine-
OH
AMP-Lys-ligase complex
Activated
Phosphorylating
complex
OH
O
O
P
O
O
NH2
N
N
N
O
N
O-PO CH2
O
HO
OH
NH2-Lysine-
O
OH
O
P
O
O
NH2-Lysine-
P
+ AMP
The formation of phosphodiester bound
ATP is required for the ligase reaction.
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, 14-16 °C, over night (o/n)
Thinking: Why the temperature is the critical factor affecting ligation?
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 2025°C.
Joining of stick ends and blunt ends
Use of linkers:
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.
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.
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, 1991-1994)
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
PCR products inserts to ligate efficiently with the vector.
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.
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.
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)
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’
The Klenow fragment is a large protein fragment
produced when DNA pol I from E. coli is enzymatically
cleaved by the protease subtilisin.
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 researchbased 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
2.3.2 T4 and T7 DNA polymerase
Source: Bacterophageies of E.coli
T4 DNA polymerase
Function：Similar to Klenow fagment
Template dependent
ＤNA polymerase
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.
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.
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
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.
2.3.3 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 75-80 ℃, 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
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 ？
2.3.4 Reverse Transcriptase
A reverse transcriptase, also known as RNAdependent 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.
Functions:
RNA-dependent DNA ploymerase;
RNase H activity: removing RNA from the RNADNA hybrids.
DNA polymerase activity
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.
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.
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).
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.
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
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
O
OH
O
OH
P
O
O
P
+ AMP
EcoRI
HO
P
P
OH
GAATTC
CTTAAG
HO
P
EcoRI
5’
P
OH
OH
P 5’
5’-pAATTC
OH
OH
OH P
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:
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.
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
DNase I in DNA foot-printing
DNase I in DNA foot-printing
An end labeled DNA probe is
incubated with a purified DNAbinding 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).
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;
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 noncoding RNAs that function in varied cellular processes.
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).
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.
2.5 Nucleic probes
Nucleic probe: In molecular biology, a hybridization
probe is a fragment of DNA or RNA of variable length
(usually 100-1000 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.
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.
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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.
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.
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.
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)