Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
The genetic material must produce a large number of copies of itself during the life cycle of an organism. The process by which a DNA molecule makes its identical copies is called DNA replication. The DNA molecule that undergoes replication may be termed as ‘parent molecule or template molecule, while the two molecules produced by replication may be called progeny molecules or daughter molecules.
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.
Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
The genetic material must produce a large number of copies of itself during the life cycle of an organism. The process by which a DNA molecule makes its identical copies is called DNA replication. The DNA molecule that undergoes replication may be termed as ‘parent molecule or template molecule, while the two molecules produced by replication may be called progeny molecules or daughter molecules.
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.
Organisms that are genetically identical are clones
Asexual Reproduction always produces clones
Laboratory Techniques have been developed that have allowed this to happen in Animals
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.
Hello everyone, I am Dr. Ujwalkumar Trivedi, Head of Biotechnology Department at Marwadi University Rajkot. I teach Molecular Biology to the students of M.Sc. Microbiology and Biotechnology.
The current presentation talks about replication and partition mechanism of plasmid. The later part of the presentation describes "Theta Model" and "Rolling Circle Model" of replication.
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
Microbial genetics is a subject area within microbiology and genetic engineering. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes
Restriction Endonuclease: The Molecular Scissor of DNA - By RIKI NATHRIKI NATH
restriction enducleases are called the molecular scissors of DNA. types of restriction enzymes, their structures, subunits, most importantly the use of Type II restriction endonuclease in recombinant technology, mechanism of enzyme action and their applications.
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.
Organisms that are genetically identical are clones
Asexual Reproduction always produces clones
Laboratory Techniques have been developed that have allowed this to happen in Animals
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.
Hello everyone, I am Dr. Ujwalkumar Trivedi, Head of Biotechnology Department at Marwadi University Rajkot. I teach Molecular Biology to the students of M.Sc. Microbiology and Biotechnology.
The current presentation talks about replication and partition mechanism of plasmid. The later part of the presentation describes "Theta Model" and "Rolling Circle Model" of replication.
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
Microbial genetics is a subject area within microbiology and genetic engineering. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes
Restriction Endonuclease: The Molecular Scissor of DNA - By RIKI NATHRIKI NATH
restriction enducleases are called the molecular scissors of DNA. types of restriction enzymes, their structures, subunits, most importantly the use of Type II restriction endonuclease in recombinant technology, mechanism of enzyme action and their applications.
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.
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
Namrata singh -recombinant dna technologyNamrata Singh
Recombinant DNA technology ( also known as genetic engineering) is the set of techniques that enable the DNA to be identified, isolated and recombined so that new characteristics can be introduced into the genome of organism. It was largely the work of Paul Berg, Herbert W. Boyer and Stanley N Cohen, although many other scientists made important contributions to the new technology as well.One important aspect of Recombinant DNA Technology is DNA Cloning.
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3. • A Restriction Enzyme or Restriction Endonuclease is a bacterial enzyme that cuts dsDNA into
fragments after recognition specific nucleotide sequence known as recognition or restriction site.
• Restriction enzymes are produced by bacteria as a defense mechanism against infection by
viruses. They restrict or prevent viral infection by degrading the DNA of invading viruses.
• The term restriction comes from the fact that these enzymes restrict the entry of foreign DNA in
the bacteria.
• Restriction enzymes, are believed to be evolved by bacteria to resist viral attack.
Restriction Enzyme
Introduction
History
• The existence of restriction enzymes was first postulated by W. Arber.
• He noticed that when the DNA of a bacteriophage entered a host bacterium it was cut up into
smaller pieces and, for this, he theorized the presence of restriction enzyme.
• In 1970, Hamilton Smith and his co-workers first isolated a restriction enzyme from the
bacterium Haemophi/us influenzae strain Rd.
• The enzyme, called Hindll, recognizes a six base-pair dsDNA sequence. After discovery of
Hindll restriction enzyme, EcoRI, was isolated and characterized from Escherichia coli strain
4. Nomenclature
The name of any restriction endonuclease consists of three parts:
1. An abbreviation of the genus and species of the organism to three letters, e.g. Eco for
Escherichia coli identified by the first letter of the genus and the first two letters of the species.
2. A letter, number or combination of the two to indicate the strain of the relevant species.
3. A Roman numeral to indicate the order in which different restriction modification systems were
found in the same organism or strain.
For example, the name of the EcoRI restriction enzyme was derived as:
E Escherichia (genus)
co coli (species)
R RY13 (strain)
I First identified (order of identification in the bacterium)
5. Restriction Site
• A restriction enzyme recognizes and binds to DNA at a specific nucleotide sequence called a
restriction site
• The enzyme then cuts both strands of the DNA within that sequence by cleaving the
phosphodiester backbone of DNA.
• Most recognition sequences exhibit a form of symmetry described as a palindrome: the
nucleotide sequence reads the same on both strands of the DNA when read in the 5’ to 3’
direction.
WAS IT A RAT I SAW
राधा की बूनी में नीबू की धारा
• The position at which the restriction enzymes cuts is usually shown by the symbol ‘/’.
• Enzymes such as EcoRI and HindIII make offset cuts in the DNA strands, thus producing
fragments with single-stranded overhanging ends called cohesive ends, while others such as
AluI and BalI cut both strands at the same nucleotide pair, producing DNA fragments with
double stranded ends called blunt-end fragments.
6.
7. Star Activity
Under non-standard reaction conditions
(such as low ionic strength, high pH), some
restriction enzymes are capable of cleaving
sequences which are similar but not
identical to their defined recognition
sequence. This altered specificity has been
termed star activity.
• Isoschizomers are pairs of R.E. That recognize the same recognition sequence and cut in same
location. For example, Sphl and Bbul (CGTAC/G) are isoschizomers of each other.
• Neoschizomers are enzymes that recognize the same sequence, but cut it differently. For
example, Smal (GGG/CCC) and XmaI (G/GGCCC) are neoschizomers of each other.
• Isocaudomers are enzymes that recognize slightly different sequence, but produce the same
ends. For example, both Sau3A (N/GATC) and BamHl (G/GATCC), gives a 5’-GATC-3’ sticky
end and have different recognition sequence.
8. Frequency of recognition sites
If it is assumed that the nucleotides are ordered in a random fashion and that the
four different nucleotides are present in equal proportion then the recognition site
of 6 bp will occur at a frequency of (¼)6 i.e. 1/4096 bases.
So, EcoRl, which recognize the sequence of 6bp(GAATTC).
If the GC content of the genome DNA is supposed to be 40%, then AT will be 60%.
Hence, G and C will be 20% each while A and T will be 30% each. For restriction
site AAGCTT, the probability of occurrence will be
3/10 × 3/10 × 2/10 × 2/10 × 3/10 × 3/10 = 324/1000000 =
~1/3086
9. References
1. Life sciences Fundamentals and Practice ll, Edition 6th , Pathfinder
publication.
2. Concept of genetics Tenth edition, klug, Cummins, et al.
Thank You