Restriction enzymes are molecular scissors found in bacteria that cut DNA molecules at specific recognition sequences. They serve as a defensive mechanism for bacteria against bacteriophages by cleaving the phage DNA. There are over 3000 known restriction enzymes that are classified into four main types based on their composition, cofactors, and cutting mechanisms. Restriction enzymes are important tools in biotechnology for manipulating DNA sequences through cutting DNA fragments with specific sticky or blunt ends, which can then be recombined through techniques like cloning.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Also referred to as Restriction Endonucleases
Molecular scissors that cut double stranded DNA molecules at specific points.
Found naturally in a wide variety of prokaryotes
An important tool for manipulating DNA.
Enters and recognizes a certain sequence on a double helix strand of DNA, usually 4-6 base-pairs long, and cuts it.
Precise by cutting both strands in same location though strands move in reverse directions; REs are able to depict the precise spot to cut
Joining together of DNA molecules from two
different species that are inserted into a host
organism to produce new genetic
combinations (i.e recombinant DNA) that are
of value to science, medicine, agriculture and
industry
RESTRICTION
ENDONUCLEASES AND
OTHER ENZYMES USED
IN GENETIC
ENGINEERING
• Also called restriction enzymes or molecular
scissors
• They are enzymes that cut DNA at or near specific
recognition nucleotide sequences known as
restriction sites
• They are found in bacteria and archaea
• A bacterium uses a restriction enzyme to defend
against bacterial viruses called bacteriophages or
phages.
• When a phage infects a bacterium, it inserts its DNA
into the bacterial cell so that it might be replicated.
Restriction enzyme prevents replication of the phage
DNA by cutting it into many pieces
• The bacterial DNA is prevented from the action of the
restriction enzyme by another set of enzymes known
as DNA methyltransferases or methylases
• DNA methylase is synthesized by the bacteria. It adds
methyl to the DNA sequence of the bacteria for
protection against restriction enzyme
• The combination of restriction endonuclease and
methylase is called RESTRICTION-MODIFICATION
SYSTEM
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
in gene cloning technique the cutting of DNA is essential. With the help of restriction endonuclease, it has been done. It also describes the restriction digest of a DNA molecule.
Restriction enzyme, also called restriction endonuclease, a protein that cleaves DNA at specific sites. It is used to cleave foreign DNA, thus eliminating infecting organisms.
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Assignment on Recombinant DNA Technology and Gene Therapy Basic principles of recombinant DNA technology-Restriction enzymes, various types of vectors, Applications of recombinant DNA technology. Gene therapy- Various types of gene transfer techniques, clinical applications and recent advances in gene therapy
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2. What are restriction enzymes?
• Molecular scissors that cut double stranded
DNA molecules at specific points.
• Found naturally in a wide variety of
prokaryotes
• An important tool for manipulating DNA.
3. Biological Role
• Most bacteria use Restriction Enzymes as a
defence against bacteriophages.
• Restriction enzymes prevent the replication of
the phage by cleaving its DNA at specific sites.
• The host DNA is protected by Methylases which
add methyl groups to adenine or cytosine bases
within the recognition site thereby modifying the
site and protecting the DNA.
5. History Of Restriction Enzyme
• First restriction enzyme was isoltaed in 1970 by
Hindll.
• He also done the subsequent discovery and
characterization of numerous restriction
endonucleases.
• From then Over 3000 restriction enzymes have
been studied in detail, and more than 600 of
these are available commercially and are
routinely used for DNA modification and
manipulation in laboratories.
6. Mechanism of Action
Restriction Endonuclease scan the length of the DNA
, binds to the DNA molecule when it recognizes a
specific sequence and makes one cut in each of the
sugar phosphate backbones of the double helix – by
hydrolyzing the phoshphodiester bond.
Specifically,the bond between the 3’ O atom and the
P atom is broken.
7. Direct hydrolysis by nucleophilic attack at the
phosphorous atom
3’OH and 5’ PO4
3- is produced. Mg2+ is required for
the catalytic activity of the enzyme. It holds the
water molecule in a position where it can attack the
phosphoryl group and also helps polarize the water
molecule towards deprotonation .
8. Palindrome Sequences
• The mirror like palindrome in which the same forward
and backwards are on a single strand of DNA strand, as
in GTAATG
• The Inverted repeat palindromes is also a sequence that
reads the same forward and backwards, but the forward
and backward sequences are found in complementary
DNA strands (GTATAC being complementary to CATATG)
• Inverted repeat palindromes are more common and
have greater biological importance than mirror- like
palindromes.
11. Blunt ends
• Some restriction enzymes cut DNA at opposite base
• They leave blunt ended DNA fragments
• These blunt ended fragments can be joined to any other
DNA fragment with blunt ends.
• Enzymes useful for certain types of DNA cloning experiments
.
12. Sticky ends
• Most restriction enzymes make staggered
cuts
• Staggered cuts produce single stranded
“sticky-ends”
13. “Sticky Ends” Are Useful
DNA fragments with complimentary sticky ends
can be combined to create new molecules which
allows the creation and manipulation of DNA
sequences from different sources.
14. ISOSCHIZOMERS & NEOSCHIZOMERS
• Restriction enzymes that have the same recognition
sequence as well as the same cleavage site are Isoschizomers
• Restriction enzymes that have the same recognition
sequence but cleave the DNA at a different site within that
sequence are Neoshizomers
Eg: SmaI and XmaI
C C C G G G C C C G G G
G G G C C C G G G C C C
Xma I Sma I
15. NOMENCLATURE OF RESTRICTION ENZYME
• Each enzyme is named after the bacterium from
which it was isolated using a naming system based
on bacterial genus, species and strain.
For e.g EcoRI
Derivation of the EcoRI name
Abbreviation Meaning Description
E Escherichia genus
co coli species
R RY13 strain
I First identified
order of identification
in the bacterium
16. TYPES OF RESTRICTION ENZYMES
• Restriction endonucleases are categorized into
three general groups.
• Type I
• Type II
• Type III
17. • These types are categorization based on:
Their composition.
Enzyme co-factor requirement.
the nature of their target sequence.
position of their DNA cleavage site relative to
the target sequence.
18. Type I
• Capable of both restriction and modification
activities
• The co factors S-Adenosyl Methionine(AdoMet), ATP,
and mg+are required for their full activity
• Contain:
two R(restriction) subunits
two M(methylation) subunits
one S(specifity) subunits
• Cleave DNA at random length from recognition sites
19. Type II
• These are the most commonly available and used
restriction enzymes
• They are composed of only one subunit.
• Their recognition sites are usually undivided and
palindromic and 4-8 nucleotides in length,
• they recognize and cleave DNA at the same site.
• They do not use ATP for their activity
• they usually require only Mg2+ as a cofactor.
20. Type III
• Type III restriction enzymes ) recognize two separate
non-palindromic sequences that are inversely
oriented.
• They cut DNA about 20-30 base pairs after the
recognition site.
• These enzymes contain more than one subunit.
• And require AdoMet and ATP cofactors for their roles
in DNA methylation and restriction
21. Type IV
• Cleave only normal and modified DNA
(methylated, hydroxymethylated and glucosyl-
hydroxymethylated bases).
• Recognition sequences have not been well
defined
• Cleavage takes place ~30 bp away from one of
the sites
22. APPLICATION OF RESTRICTION ENZYMES
• They are used in gene cloning and protein expression
experiments.
• Restriction enzymes are used in biotechnology to cut
DNA into smaller strands in order to study fragment
length differences among individuals (Restriction
Fragment Length Polymorphism – RFLP).
• Each of these methods depends on the use of
agarose gel electrophoresis for separation of the
DNA fragments.
23. RFLP is a difference in homologous
DNA sequences that can be detected by the
presence of fragments of different lengths after
digestion of the DNA samples.
What is RFLP
24. The method of analysis of DNA by RFLP involves
the following steps:
1- In the first step fragmentation of a sample of
DNA is done by a restriction enzyme, which can
recognize and cut DNA wherever a specific
short sequence occurs, in a process known as a
restriction digest.
Method of DNA analysis by RFLP
25. 2- The resulting DNA fragments are then separated by
length through a process known as agarose gel
electrophoresis.
3- Then transferred to a membrane via the Southern blot
procedure.
4- Hybridization of the membrane to a labeled DNA
probe will done and then determines the length of the
fragments which are complementary to the probe.
26. 5- Then we will observe the fragments of different
length.
An RFLP occurs when the length of a detected
fragment varies between individuals. Each fragment
length is considered an allele, and can be used in
genetic analysis.