PAPER 0703

Basics Concepts and Techniques in Molecular

Biology

Lecture 1
(Semester I - 2015)
 Recombinant DNA Technology
Bacterium Plasmid Cell containing gene
isolated of interest

DNA
isolated
Gene
Bacterial inserted
Plasmid into plasmid
chromosome
Gene of
Recombinant DNA interest DNA
(plasmid)

Plasmid put into

bacterial cell

Recombinant
bacterium

Cell multiplies with

gene of interest

Copies of gene Copies of protein

Gene for pest Clones of cell Protein used to

resistance make snow form
inserted into at higher
plants temperature

Gene used to alter bacteria Protein used to dissolve blood

for cleaning up toxic waste clots in heart attack therapy
 Recombinant DNA Technology

rDNA: An artificially created DNA molecule which combines DNA sequences not
usually found together in nature.

Gene Manipulation: Technique involved in creation of rDNA.

rDNA technology has revolutionized the research in the field of medicine

and agriculture.
 Escherichia coli : A Key Laboratory Organism in RDT

 Popular model system for molecular geneticist due to availability of mutants,

Understanding of gene regulation and several isolated plasmids.

 First cloning experiment in E. coli.

 Played dominant role in the early years of rDNA era.

 Most widely used cloning host today due to ease of handling and manipulation.

 Cloning techniques extended to other microorganisms such as Bacillus subtilis,

Pseudomonas sps., yeast , filamentous fungi and even higher eukaryotes.
Genome Sequencing and rDNA Technology
 RDT and Genomics: Foundation of Biotechnology Industry

 Sequencing information on the genomes of several organisms has provided

opportunities for manipulation of genes and their usage in medicine and
agriculture.

 Parallel development in the field of bioinformatics facilitated the new

sequencing strategies.

 Comparative genomics has been useful in studying evolution.

 Genome sequencing projects became international collaborative projects and

Promoted proliferation of scientific interactions.
Recombinant DNA Technology: A Roadmap
Principles and Tools of Recombinant DNA Technology

Requirements for recombinant DNA technology:

1. Enzymes

2. Vector System

3. Gene of Interest

4. Cloning system

5. Experimental Organism
Cloning Components and Methods

Conventional Methods by Restriction Digestion

PCR-based cloning

Adaptors and Linkers

TA cloning

Homopolymer tailing

Topo Cloning

Recombination-based Gateway Technology

 Molecular Biology Enzymes

Types of enzymes employed in rDNA technology and molecular biology:

• Nucleases

• Ligases

• Polymerases

• Modifying enzymes
Enzymes Useful in Recombinant DNA Technology

 Nucleic Acid Manipulating Enzymes

 Nucleases
 deoxyribonucleases
 ribonucleases

 Ligases

 Polymerases
Terminal Deoxynucleotidyl transferase

 Nucleic Acid Modifying

Enzymes

 Alkaline Phosphatase

 Polynucleotide Kinase
 NUCLEASES

EXONUCLEASES ENDONUCLEASES
(removes nucleotides one at a time (breaks phosphodiester bonds within a DNA
from the end) molecule)
 Types & Properties of Nucleases
Activity
    on      
ENZYME TYPE ssDNA dsDNA ssRNA dsRNA
           

Bal31 Nuclease 3'----> 5' exonuclease Yes Yes Inefficient  

  endonuclease Yes No Inefficient  

           
Dnase 1 endonuclease Yes Yes No No
           
Exonuclease I 3'---> 5' exonuclease Yes No No No
           
Exonuclease III 3'---> 5' exonuclease No Yes No No
DNA-RNA
  endonuclase ~ ~ hybrid  
           
Exonuclease V 3'---> 5' exonuclease Yes Yes No No
  5'---> 3' exonuclease        
           
ExonucleaseVII 3'---> 5' exonuclease Yes No No No
  5'---> 3' exonuclease        
           
Lambda exonuclease 5'---> 3' exonuclease No Yes No No
           
Mung Bean nuclease Endonuclease Yes No Yes No
           
P1 nuclease exonuclease Yes Yes Yes Yes
  endonuclease Yes Yes Yes Yes
           
Types & Properties of Nucleases (contd.)
 Nucleases Active on Both DNA and RNA
Enzyme Substrat Products Applications Heat
e Inactivation

Terminator™ 5´- ssDNA or dNMPs or Removal of 5´- 10 min, 65°C

Phosphate- ssRNA NMPs monophosphorylated DNA or
Dependent primers or oligos. Enrichment of
Exonuclease ssDNA or ssRNA molecules lacking
5´-monophosphate groups
Endonuclease ssDNA, di-, tri-, and Removal of DNA and RNA from No
dsDNA, tetra- protein preps. Removal of host
or RNA nucleotides DNA from phage preps
 DNA Endonucleases
Enzyme Substrate Activity Products Applications Heat
Inactivationa
RNase-Free DNase I          
(bovine pancreas) dsDNA and Activated by divalent cations. In Oligos and Removing DNA from RNA preps. 20 min, 75°C
ssDNA presence of Mg2+, it cleaves each dNMPs with 5´ Random nicking of dsDNA. DNase
DNA strand randomly and phosphate and footprinting
independently, preferentially 3´-OH
adjacent to pyrimidines. In presence
of Mn2+, it cleaves both strands
simultaneously, generating
fragments with blunt ends of 1-2-
base overhangs
Baseline-ZERO™ dsDNA and Digests dsDNA or ssDNA down to Mononucleoti Removing DNA from RNA preps 20 min, 75°C
DNase ssDNA mononucleotides des
Endonuclease IV, E. dsDNA Cleaves sugar-phosphate bond 5´ of dsDNA with DNA repair and antitumor drug NT
coli with an an abasic site single- research. Base Excision Sequence
abasic site nucleotide Scanning of DNA containing dUMPs
gaps. The
cleaved ssDNA
strand has a 3
´-OH
T4 Endonuclease V UV- First cleaves N-glycosidic bond 5´ of Nicked dsDNA Research on repair of DNA exposed to NT
irradiated thymine dimers, then cleaves sugar- with an abasic UV light
DNA with phosphate bond 3´ of the abasic site site at the 3´
thymine end of the cut
dimers and thymine-
dimer bases at
the 5´ end of
the cut
Lambda Terminase dsDNA Cleaves both strands at 5´ ends with Rapid sizing or restriction mapping of NT
with cos bacteriophage lambda cos sites overhangs 12 BAC, fosmid, or cosmid clones
sites bases in length
 DNA Exonucleases
Enzyme Substrate Activity Products Applications Heat
Inactivation
Removal of ssDNA and
Exonuclease I, E. coli ssDNA 3´→5´ exonuclease dNMPs oligonucleotides. 15 min, 80°C

dNMPs and
ssDNA on the
opposite
3´→5´ exonuclease that digests strand. Partial Used with S1 Nuclease or Mung Bean
duplex DNA from the 3´ end of a nick digestion Nuclease to make nested deletions.
or a blunt or 3´-recessed end; Exo III produces Preparation of ssDNA templates for
also has 3´-DNA phosphatase and dsDNA having sequencing. Site-directed mutagenesis.
apurinic DNA endonuclease 5´ extensions Preparation of labeled strand-specific
Exonuclease III, E. coli dsDNA activities. of ssDNA. probes. 20 min, 70°C
Exonuclease that digests in both 5 Removal of primers and single-stranded
Exonuclease VII ssDNA ´→3´ and 3´→5´ directions. dNMPs oligos. 10 min, 95°C
linear Selectively digests linear DNA. No Remove chromosomal DNA fragments
Plasmid-Safe™ ATP- ssDNA and activity on nicked or closed-circular from plasmid, fosmid, and BAC
Dependent DNase dsDNA DNA. dNMPs preparations. NT

dNMPs and
ssDNA on the
opposite
strand. Partial
5´→3´ exonuclease that digests digestion
dsDNA from 5´-phosphorylated blunt produces
or recessed ends. It has low activity dsDNA having
on 5´-hydroxyl ends and is not active 3´ extensions Preparation of ssDNA templates for
Lambda Exonuclease dsDNA on nicked DNA. of ssDNA. sequencing. 10 min, 75°C
 DNA Exonucleases (contd.)
An ATP- and Mg2+-dependent
exonuclease that digests linear DNA
in both 5´→3´ and 3´→5´ directions.
RecBCD Nuclease, E. dsDNA and Not active on nicked or closed- Removal of linear DNA from circular
coli ssDNA circular dsDNA. dNMPs DNA. NT
5´→3´ exonuclease that digests
ssDNA with a 5´ phosphate or a 5 Removal of primers and ssDNA from
Rec J Exonuclease ssDNA ´OH. dNMPs dsDNA. 20 min, 65°C
5´→3´ exonuclease that also has dNMPs.
single-strand-specific endonuclease Circular ssDNA
activity in presence of 1-10 mM is obtained
Mg2+ ions. At <1 mM Mg2+, the 5 from closed- Plasmid mutagenesis. Oligonucleotide
´→3´ exonuclease can digest from a circular dsDNA site-directed mutagenesis. Removing
dsDNA and nick in closed-circular dsDNA without in presence of linear DNA from plasmid preps.
T5 Exonuclease ssDNA digesting the opposite strand. <1mM Mg2+. Preparation of circular ssDNA NT
 RNA Endonucleases
Enzyme Substrate Activity Products Applications Heat
Inactivationa
Oligoribonucle
otides with 3´-
cytidine or 3´- Removal of RNA from DNA preps.
Cleaves ssRNA 3´ of pyrimidine uridine RNase protection assays. RNA mapping
RNase A ssRNA residues. residues and structure studies. NT
NMPs with 5´-
OH and 2´,3´- Removal of RNA from DNA preps.
cyclic RNase protection assays. Mismatch
Cleaves ssRNA between all monophospha detection of single basepairs in
RNase I, E. coli ssRNA dinucleotide pairs. te RNA:RNA or RNA:DNA hybrids. 15 min, 70°C
dsRNA with 5
Cleaves dsRNA in presence of Mg2+ ´phosphate Random cleavage of long dsRNA to
to 12-15-bp dsRNA. Cleaves dsRNA in and two-base short dsRNA. RNA interference (RNAi).
presence of 20mM Co2+ or Mn2+ to 3´ overhangs Studies on RNA structure, RNA
RNase III, E. coli dsRNA 18-25-bp dsRNA. with 3´ OH processing, and maturation. No
Oligoribonucle Elimination of RNA prior to second-
RNA in Cleaves RNA in RNA:DNA hybrid otides with 5´ strand cDNA synthesis. Removal of
RNA:DNA without affecting unhybridized RNA phosphate and poly(A) tails from mRNA hybridized to
RNase H, E. coli hybrid or DNA. 3´ OH oligo(DT). 10 min, 65°C
Oligoribonucle
Hybridase™ RNA in Cleaves RNA in RNA:DNA hybrid otides with 5´
Thermostable RNase RNA:DNA without affecting unhybridized RNA phosphate and
H hybrid or DNA. 3´ OH High-stringency hybrid selection. No
Oligoribonucle
RNase T1, Aspergillis otides with 3´- RNA mapping and structure studies.
oryzae ssRNA Cleaves ssRNA 3´ or GMPs GMP residues Removal of RNA from DNA preps. NT
RiboShredder™ Removal or all RNA frm genomic and
RNase Blend ssRNA Efficiently degrades all RNA. NMPs cloned DNA preps. No
 Applications of nucleases in molecular biology

 DNase I--degradation of DNA template in transcription reactions,

genomic DNA preps

 Micrococcal nuclease—non-specific endonuclease, digest ds, ss,

Circular or linear nucleic acids. Useful in degrading nucleic acids in
Protein preps.

 Mung bean nuclease—ss-specific exonuclease, removal of 5’ or 3’

extensions from DNA or RNA termini, generation of new restriction sites
 RESTRICTION ENDONUCLEASES
 Site-specific endonucleases, also known as Molecular Scissors or Scalpels.

 Named so as they restrict or prevent viral infection by degrading the

invading nucleic acid.
RESTRICTION ENDONUCLEASES
 RESTRICTION ENDONUCLEASES

 Host DNA is protected by restriction endonucleases as it is modified by

methylases
 History & Discovery
 Luria & Human and Bertani & Weigle (1952-53)
Observed Restriction phenomenon (host-controlled or
host-induced variation)

 Arber & Dussoix (1962)

Molecular basis of restriction explained

 Meselson & Yuan (1968)

Isolated first restriction endonuclease E. coli. K (Type I)

 Smith & Wilcox (1970)

Isolated endonuclease R (Type II) i.e. Hemophilus influenzae

 Kelly & Smith (1970)

Determined the DNA sequence recognized by endonuclease R.

 Danna and Nathans (1971)

Endonuclease R cleaves specific DNA sequences in SV40
 Hamilton O. Smith Daniel Nathans Werner Arber

The Nobel Prize in Physiology or Medicine 1978 was

awarded jointly to Werner Arber, Daniel Nathans and
Hamilton O. Smith "for the discovery of restriction enzymes
and their application to problems of molecular genetics".
 Diversity of Restriction endonucleases

 Primarily found in bacterial genomes and plasmids.

 Also found in archaea, viruses and eukaryotes.
 Certain bacterial strains are rich source more than one restriction
enzymes. Eg. Neisseria and Helicobacter pylori
 >3500 restriction enzymes that recognize 259 different DNA sequences
are known (Williams R. J., 2003), majority of these are Type II.
 Nomenclature of Restriction endonucleases

 Naming of enzyme is as follows:

 first letter: genus name, italicized and capitalized

 second two letters: species name, italicized and not capitalized
 Other variant designations follow: designates particular strain
 Roman numeral identify multiple enzymes from the same
bacterium

 Example : EcoR I
Number of the enzyme
Escherichia coli Strain RY13
 Classification of Restriction endonucleases

 Classified on the basis of

 Structure or composition
 recognition site and cleavage site
 Cofactor & activator requirement

 4 Types : Type I, Type II, Type III and Type IV

 All restriction endonucleases have Restriction, Methylase and

Specificity domains present on same or different polypeptides
 Different Classes of Restriction Endonucleases
Type Subunit Cofactors & Recognition site Cleavage site
structure activators

I One enzyme with Mg+2 Interrupted Bipartite Distant (>1000 bp) and variable from
(First) three subunits, AdoMet (SAM), (asymmetrical; one recognition site
each for ATP containing 3 nt and EcoKI:
EcoKI:
recognition, other with 4-5 nt AAC(N6)GTGC(N>400) ↓
cleavage & separated by a TTG(N6)CACG(N>400)↑
methylation spacer of about 6-8
(RMS-HsdM, nt) Interact with two asymmetrical bi-
HsdR & partite recognition site, translocate
HsdS(HsdM2Hsd the DNA in an ATP-hydrolysis
R2HsdS- dependent manner and cut DNA
structure of distal to recognition site (approx.
active enzyme)) half-way between two sites)
II Simple subunit Mg+2 (with one Palindromic, Defined, within recognition site, may
(Most organization. exception) undivided, 4-8 nt in result in a cohesive (3' overhang or
commonly Usually length 5'overhang) or blunt end
used) homodimeric or EcoRI:
EcoRI:
homotertrameric G↓A A T T C
enzymes C T T A A↑G

Two different
enzymes, one for
recognition
(Homodimer) &
other for
methylation
(monomer)
 Different Classes of Restriction Endonucleases

III One enzyme with Require Mg+2, Non Palindromic Cuts approx 25 bases downstream to
2 different ATP recognition site
subunits, one for EcoP15I:
recognition and Stimulated by CAGCAG(N) 25–26↓
modification AdoMet GTCGTC(N) 25–26↑
(Mod) and other
for cleavage Interact with head-to-head
(Res) Mod2Res2 arranged asymmetrical recognition
stoichiometry of sites, translocate DNA in an ATP-
active active hydrolysis dependent manner and
nuclease cut the DNA close to one
recognition site.

IV Heterotrimer 6 Mg+2 Interrupted Cuts both strands on both sides of

(2 R-M, 1 S) AdoMet, GTP Bipartite recognition site a defined, symmetric,
(two separated non- short distance away and leaves
palindromic 3' overhangs, for example, BcgI:
BcgI:
sequences that are ↓10(N)CGA(N)6TCG(N)12↓
inversely oriented) ↑12(N)GCT(N)6ACG(N)10
↑12(N)GCT(N)6ACG(N)10↑ ↑

Generally recognize and cleave

methylated DNA. McrBC is widely
used enzyme.
 Characteristic Properties of Type II Class of REs

 Most commonly found

 Highly specific cleavage of DNA strands

 Recognize palindromic sequences

(rotational dyad symmetry)

5’-----G AATT C----3’

3’-----C TTAA G----5’

 Very useful in recombinant DNA research

 Type II has subclasses on the basis of their properties :

Type II a : recognize asymmetric sequences

Type II b : cleave DNA at both sides of the recognition
sequence
Type II c : has both cleavage and modification domains
within one polypeptide.
Type II e : need to interact with two copies of their
recognition sequence for efficient cleavage; one copy
being the target for cleavage and the other serves as an
allosteric effector.
Type II f, II h, II m, II s, II t ………………
 Recognition sites are usually:

4 bases long- BfaI, AluI, HpaII

6 bases long- EcoRI, BamHI, PvuII
8 bases long- NotI, PmeI, SbfI

 What do you expect if you cut your plasmid DNA with

• 4 bp cutter ?

• 6 bp cutter ?

• 8 bp cutter ?
Characteristic Properties of Type II Class of REs (contd.)

 Generates sticky or blunt ends

Sma I

 Different kinds of blunt ends can be ligated together. Some ligations

may lead to generation of new cleavage sites.

CCCGGG GATATC C CCATC

+
GGGCCC CTATAG GGGTAG

GATATC + TCGCGA GATCGA or GATCGA

(EcoRV) (NruI) (MboI) (TaqI)
Characteristic Properties of Type II Class of REs (contd.)

 Generates sticky or blunt ends

Hind III

5’----G AATT C---3’ 5’----C TGCA G ---3’

3’----C TTAA G---5’ 3’----G ACGT C ---5’

5’ overhangs 3’ overhangs
(EcoRI) (PstI)

 Overhanging ends can anneal through complementary hydrogen

bonding
Restriction sites many contain sites for other enzymes
within them.
 Cohesive ends can be made into blunt ends.

 5’ overhang is filled in or flushed: 3’OH end becomes the

pimer for the extension through addition of bases by DNA
polymerases, thus filling recessed end converting ss
overhang into ds DNA.

 3’ overhang is flushed : use of an exonuclease.

 Generation of New Restriction Sites

 New restriction sites may be generated by:

blunt-blunt ligation

blunt-blunt

filled-in 5’ overhangs to generate blunt ends

compatible cohesive ends

Blunt-blunt Ligation resulting in recleavable blunt ends.

GAT^ATC TCG^CGA
CTA^TAG AGC^GCT

EcoRV NruI

GAT.CGA
CTA.GCT
Ligated Product

^GATC T^CGA
CTAG^ AGC^T
MboI TaqI
Filled-in 5’ overhangs to generate blunt ends
Compatible Cohesive Ends Can be Ligated and Recleaved.

 Different restriction endonucleases may produce sticky

ends that are compatible or can anneal.

 joined sites may not always recreate recognition

sequences for either of the enzyme

 Joined sequences may sometimes create a site for a

new enzyme (eg. by filling in the overhangs or
compatible overhangs)
 Compatible Cohesive Ends Can be Ligated

1. UNCLEAVABLE:
 Compatible Cohesive Ends Can be Ligated

2. RECLEAVABLE:
BamHI BglII
G GATCT
CCTAG A

GGATCT
CCTAGA

Not cleaved by BamHI or BglII

Cleaved by DpnII (^GATC)

BamHI BstYI
G GATCY
CCTAG R

GGATCY
CCTAGR

Can be cleaved by BamHI if Y=C

Can be cleaved by BstYI

Cleaved by DpnII (^GATC)

Y= C or T
R= A or G
 Characteristic Properties of Type II Class of REs (contd.)

 Different enzymes that recognize the same sequence- Isoschizomers

HpaII and MspI cleave as: C^CGG

Sph I and BbuI cleave as: GCATG^C

 Subset of isoschizomers that recognize the same sequence, but cleave at

different positions from the prototype. Different enzymes cleave the same site at
different position - Neoisoschizomers

SmaI: CCC^GGG AatII: GACGT^C

XmaI: C^CCGGG ZraI: GAC^GTC

 Characteristic Properties of Type II Class of REs (contd.)

 All restriction enzymes require

 Mg2+ cofactor

 generate 5’-PO4 and 3’-OH ends

 require optimal salt concentrations

 some enzymes exhibit star activity or off-target activity:

loss of fidelity/relaxed or altered specificity under extreme or non-standard
conditions. Enzyme cleaves similar but not identical sequences (non-
canonical sequences). This includes single base substitutions, truncation of
the outer bases in the recognition seq.

Altered specificity depends largely on the enzyme and the reaction

conditions which induce star activity.

Examples are: BamHI, EcoRI, SalI, PvuII, SspI

Star Activity
 Hi-Fidelity Restriction Enzymes

 Improved restriction enzymes.

Engineered (mutations introduced) to have high specificty and reduced star

activity.
Eg. SalI requires Buffer 3 (high ionic strength) but SalI-HF can work well in
Buffer 2 and 4 (moderate ionic strength). This means that star activity won’t be a
problem in case you use these buffers (which otherwise were non-optimal for
native enzyme).

Moreover most of HF enzymes work in one buffer (CutSmart buffer in case of

company NEB). Therefore, their are well-suited for double digestions.

Provide more flexibility with setting up of cloning experiments.

Characteristic Properties of Type II Class of REs (contd.)

 linear versus coiled DNA: cccDNA requires 3-5 fold more enzyme
than linear DNA.

 G+C content of a DNA molecule: some restriction endonucleases

preferentially cleave certain sites faster than the other sites spread
in the other portions of genome.

Example: Plasmid pBR322 has 4 recognition sites for NarI. It rapidly

cleaves two of the four sites but seldom the other two.

 Digestion of sites near termini requires 4-8 bases for efficient

digestion.

Example: SalI cleaves G^TCGAC. If GTCGAC-------cleavage is 10%

ccggatGTCGAC--------cleavage is
90%
 Mechanism of Type II Class of REs

on-specific binding and interaction with phosphate

ackbone (loosely bound , open state)

near diffusion or sliding of the enzyme

coRI-scans 2x106 bp @ 1.7x106 bp/sec)

ecognition of target site, formation of H-bonds with

cognition site bases in the presence of Mg 2+. Additional
rmation of Van der Waals base contacts and H bonds to the
ackbone. (Partially bound, closed state)

onformational changes in enzyme and DNA & expulsion of

ater molecules for intimate contact. Activation of catalytic
enter. Flanking sequences may influence specific binding of
nzyme to the recognition site. (Tightly bound, cleavage ready
osed state)

eavage of phosphodiester bond in each strand and release

the product. Cohesive or blunt end products are released
epending upon the enzyme and recognition site.
TARGET SITE LOCATION:
•Open DNA binding site is required.

•Face problem in finding specific site due to the

presence of huge non-specific sites on DNA.

•Facilitated diffusion is an effective process that

increases processivity of RE by a factor of 10.
Optimally RE can scan 106 bp in one binding event.
Sliding (linear or 1-D diffusion), jumping or hopping
(3-D diffusion) and intersegment transfer (only
possible for proteins with two DNA binding sites)
mechanisms account for this target site location.
Principally different but not mutually exclusive.

Question: What is the relative contribution of each

mechanism ?

RECOGNITION:
Involves conformational adaptations of protein and
DNA with water and counter-ion release at the
protein-DNA interface.
Inspection of DNA-protein co-crystal structures
concluded:
1. Blunt end or sticky with 3’ overhangs approach
minor groove while sticky with 5’ overhangs
contact major groove.
Pinguod et al., CMLS, 2005
2. Specific DNA-binding is accompanied by distortions of DNA that brings functional
groups of DNA into positions required for optimal recognition and also position the
phosphates such that they can support phosphodiester bond hydrolysis. Also
conformational changes in protein structure.

3. Formation of highly co-operative H-bond network. Additionally, van der Waals

interactions and hydrophobic contacts are formed to the bases of recognition sequence.

COUPLING BETWEEN RECOGNITION & CATALYSIS:

Least understood aspects of enzymology of Type II RE. Co-ordination of two catalytic

centres occurs. Since Type II cleave ds DNA, there is a cross-talk between amino acid
residues involved in a base-specific contact in one subunit with the catalytic centres of
both subunits.

PHOSPHODIESTER BOND HYDROLYSIS:

Preparation of the attacking nucleophile by deprotonation.

Nucleophilic attack of hydroxide ion on the phosphorus leading to formation of pentavalent

transition state.

Departure of 3’OH leaving group.

 Restriction Endonucleases and Methylases
 Methylases protect host from cleavage by corresponding restriction
endonuclease.

 Prokaryotes contain three site-specific DNA methylases:

 Dam methylase - N6 position of adenine in GATC

 Dcm methylase - C5 position of cytosine in CCAGG & CCTGG

 EcoKI methylase-adenine in sequences AAC(N6A)GTGC, GCAC(N6A)GTT

Methyltransferases tranfer methyl group from SAM to either adenine or

cytosine residues.
Methylation to be considered when designing restriction cleavage reactions
due to methylation sensitivity of these enzy,es.
Applications of Restriction Endonucleases

 Indispensable in molecular cloning techniques

 Useful in mutational analyses

 Generation of RFLPs

 Construction of restriction maps

 An important tool for detection of diseases

1. RESTRICTION MAPPING
 2. DETECTION and DIAGNOSTICS

Example 1: Detection of MSUD (Maple Syrup Urine Disease)

 MSUD is an autosomal recessive disorder of branched-chain amino acid

metabolism or ketoaciduria. Inherited disorder with symptoms as sweet-smelling urine.

Attributed to defective activity of BCKAD (branched chain alpha-keto acid dehydrogenase

complex) enzyme which is a multienzyme complex. Complex composed of E1, E2 and E3
subunits.

Mutation in second exon of E2 gene (Fisher et al., 1993) as 2 bp deletion (E2ΔAT).

Results in destruction of AflIII site in the exon.

Example 2: Detection of Sickle Cell Anemia

RFLP / MstII test for Sickle-Cell Anemia

 3. GENERATION OF RFLP
MAPS
Restriction Fragment length Polymorphism
Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-

Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-

Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC

Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC-

APPLICATIONS OF RFLP MAPPING

1. Paternity Testing.

2. Disease Status.

3. Forensics.

4. Genetic Mapping
4. GENE CLONING and RECOMBINANT DNA TECHNOLOGY
 SUGGESTED READING

 Gene Cloning- T.A. Brown

 Principles of Gene manipulation and genomics- Primrose and Twyman

 From Genes to Clones- E. L. Winnacker

 Roberts RJ. 2005. How restriction enzymes became the workhorses of molecular
biology. PNAS 102(17):5905-5908.

 Pingoud A et al. 2005. Type II restriction endonuclease : structure and mechanism.

Cell. Mol. Life Sci. 62(2005):685-707.

 Zahran et al. 2010. Mechansim of DNA recognition by the restriction enzyme, EcoRV.
J. Mol. Biol. 401:415-432.

 New Englab Biolabs Laboratory Catalog or visit www.neb.com