the cutting and joining functions in DNA manipulation. All other enzymes involved in genetic engineering fall under the broad category of enzymes known as DNA modifying enzymes. These enzymes are involved in the degradation, synthesis and alteration of the nucleic acids. They are broadly categorized as nuclease, polymerase and enzymes modifying ends of DNA molecules.
phosphodiester bonds of DNA to degrade the nucleic acids. Actually, Phosphodiester bonds holds nucleotides together. There are two nucleases, viz., i. Exonucleases- Degrade DNA from the terminal ends. ii. Endonucleases- Act on the internal phosphodiester bonds. E.g. Restriction endonucleases. Figure showing phosphodiester bond in DNA
with protruding 5’-ends. Nuclease Bal 31- complex enzyme. Its primary activity is a fast-acting 3’ exonuclease, which is coupled with a slow-acting endonuclease. When Bal 31 is present at a high concentration these activities effectively shorten DNA molecules from both termini. Some Endonucleases: Nuclease S1 specifically acts on single-stranded DNA or RNA molecules. Deoxyribonuclease I (DNase I) cuts either single or double-stranded DNA molecules at random sites.
time from an end of a polynucleotide chain. Act upon genome and digest the phosphodiester bonds of base pairs on 5’ and 3’ ends of a single stranded DNA or at single strand nicks or gaps in double stranded DNA. Types of exonucleases: a. Lambda exonuclease b. Exonuclease III c. Bal 31 Exonuclease A nick is a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides
in the 5’ →3’ direction to create long single stranded overhangs used in the recombination by the bacteriophage λ. It uses the energy release in the degradation of its DNA substrate to produce motion. It also degrades single stranded and non-phosphorylated substrates at a highly reduced rate. It removes nucleotides from 5’ ends of double stranded DNA. It used for 5’ end modification.
3’ →5’ direction but not double stranded DNA. It requires the presence of magnesium and free 3’- hydroxyl terminus for activity. It used for removal of residual single strand DNA including oligos from reaction mixture as well as nucleic acid mixture.
mononucleotides from 3-OH end of the double stranded DNA. It digests duplex DNA in a 3’ →5’ direction from a nick, a blunt or 3’-recessed end, producing single stranded DNA fragments on the opposite strand. It also extends nicks in duplex DNA to create single stranded gaps. It works actively on single stranded DNA. It is used for 3’ end modification of DNA fragments.
within a polynucleotide chain are called “endonuclease”. Few classes of endonuclease cleave only at the specific nucleotide sequences. Most widely used endonucleases are Dnase I and Rnase. They act upon genetic material and cleave the double stranded DNA at any point except the ends, but their actions involves only one strand of the duplex. Some examples: a. S1 Nuclease b. Mungbean nuclease c. Deoxyribonuclease I (DNase I) d. Ribonuclease (Rnase)
source of the enzyme. Low concentration of S1 nuclease digests single-stranded DNAs or RNAs, double stranded nucleic acids are degraded by high concentration of enzymes. Moderate concentrations can be used to digest the double stranded DNA nicks or small gaps. It degrades single stranded DNA or single strand protrusion of double stranded DNA with cohesive ends. It converts cohesive ends into blunt ends. It used to remove the incompatible ends so that over lapping ends may be developed for annealing of two fragments of DNA molecules. Commonly used to analyze the source of DNA:RNA hybrids. Used to remove single stranded extensions (overhangs) of DNA fragments to produce blunt ends. Used to remove single stranded tails from DNA molecules to create blunt ended molecules. Used to opening hairpin loops generated during synthesis of double stranded cDNA. DNA:RNA Hybrid They form during transcription when nascent RNA is in close proximity to its DNA template An RNA molecule in the process of being synthesized (hence incomplete) or a complete, newly synthesized RNA molecule before any alterations have been made
(Vigna radiata) Degrades single stranded DNA or RNA, but not double stranded. Produces mono and oligo-nucleotides carrying a 5’-terminus. Used for removal of hairpin loops during cDNA synthesis. Restriction site modification or removal by digestion of single stranded protruding ends.
or single stranded DNA. Cleavage occurs adjacent to pyrimidine (C or T) residues. The major products are 5’-phosphorylated di, tri and tetranucleotides. It hydrolyze each strand of duplex DNA independently in the presence of magnesium ions generating random cleavage. It cleaves both strands of DNA at about the same site resulting in production of blunt ends or fragments with 1-2 base overhangs. It eliminates DNA (e.g. plasmid) from preparations of RNA.
smaller components is called ribonuclease. Two groups: Endoribonucleases and Exoribonucleases Endoribonuclease: Ribonuclease A (RNase A)- Cleaves single-stranded RNA at the 3’ end of pyrimidine residue. Ribonuclease H (RNase H)- Cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex resulting in production of 3’-OH and 5’- PO4 - terminated products. Ribonuclease P (RNase P)- Cleaves off an extra or precursor sequence of RNA and tRNA molecules. Ribonuclease T1 (RNase T1)- Cleaves single stranded RNA after guanine residues on their 3’ end. Exoribonuclease: Ribonuclease D (RNase D)- Involve in structed RNA processing. Ribonuclease R (RNase R)- Important role for RNase R in mRNA decay.
catalyze the synthesis of nucleic acid molecules. Some important Polymerase: i. DNA-dependent DNA polymerase: Copies DNA from DNA. ii. DNA-dependent RNA polymerase: Produces RNA from DNA. iii. RNA-dependent DNA polymerase/Reverse transcriptase: Synthesize DNA from RNA
an RNA-dependent DNA polymerase, and therefore produces a DNA strand from an RNA template. It has no associated exonuclease activity. The enzyme is used mainly for copying mRNA molecules in the preparation of cDNA (complementary or copy DNA) for cloning, although it will also act on DNA templates. Reverse transcriptase is a key enzyme in the generation of cDNA; the enzyme is an RNA- dependent DNA polymerase, which produces a DNA copy of an mRNA molecule.
in lymphocytes. Also known as deoxynucleotidyl transferase. It has a property to add oligodeoxynucleotide tail to 3’-OH end of double stranded DNA fragments. It extends homopolymer tails this phenomenon is called as Homopolymer tailing. It catalyzes the addition of nucleotides to the 3’-OH terminus of DNA. It also works on single stranded DNA, including 3’ overhangs of double stranded DNA. It also adds homopolymers of ribonucleotides to the 3’end of DNA. Cobalt is necessary cofactor for activity of the enzyme.
by using the DNA-dependent RNA polymerase encoded by bacteriophages for in vitro transcription. The polymerases are either purified from phage infected bacteria or produced as recombinant protein. Examples: T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.
synthesis on DNA or cDNA template. It also catalyze 5’→3’ and 3’ →5’ exonucleolytic degradation of DNA. DNA polymerase I (DNA pol I) investigated by A. Kornberg in E.coli. The addition of mononucleotide to the free –OH end of DNA chain is catalyzed by this enzyme. It catalyzes the two reactions: 3’ →5’ exonuclease activity and 5’ →3’ exonuclease activity. Other DNA polymerases are DNA polymerase II and III. DNA polymerase II: Catalyzes via 3’ →5’ exonuclease activity. DNA polymerase III: Several times more active than I and II. Where there is performed DNA template it produces a parallel strand in the presence of ATP.
DNA polymerase and a 3’ →5’ exonuclease. Activities are similar to Klenow fragment. Used for producing blunt ends in DNA with 5’ or 3’ overhangs. 2.6 T4 DNA polymerase: DNA polymerase of T7 bacteriophage possesses polymerase. 3’ →5’ exonuclease activity. It can be used in DNA sequencing by the chain termination technique. 2.7 Thermostable DNA polymerase: Isolated from Thermus aquatics bacteria. Using as Taq polymerase in PCR and it catalyze template directed synthesis of DNA from nucleotide triphosphates.
reaction, where the 5’→3’ exonuclease function degrades the non-template strand as the polymerase synthesizes the new copy. The 5’→3’ exonuclease function of DNA polymerase I can be removed by cleaving the enzyme to produce what is known as the Klenow fragment. This retains the polymerase and 3’→5’ exonuclease activities. The Klenow fragment is used where a single-stranded DNA molecule needs to be copied; because the 5’→3’ exonuclease function is missing; the enzyme cannot degrade the non-template strand of dsDNA during synthesis of the new DNA. The 3’→5’ exonuclease activity is suppressed under the conditions normally used for the reaction. A modified form of DNA polymerase I called the Klenow fragment is a useful polymerase that is used widely in a number of applications. Major uses for the Klenow fragment include radiolabeling by primed synthesis and DNA sequencing by the dideoxy method in addition to the copying of single-stranded DNAs during the production of recombinants. A major use of this enzyme is in the nick translation procedure for radiolabeling DNA.
The enzymes alkaline phosphatase, polynucleotide kinase, and terminal transferase act on the termini of DNA molecules and provide important functions that are used in a variety of ways. The phosphatase and kinase enzymes, as their names suggest, are involved in the removal or addition of phosphate groups.
enzyme removes phosphate groups from the 5’ ends of DNA, leaving a 5’-OH group. The enzyme is used to prevent unwanted ligation of DNA molecules, which can be a problem in certain cloning procedures. It is also used prior to the addition of radioactive phosphate to the 5’ ends of DNAs by polynucleotide kinase.
adds nucleotides to any available 3’ terminus. Although it works best on protruding 3’ ends, conditions can be adjusted so that blunt-ended or 3’- recessed molecules may be utilized. The enzyme is mainly used to add homopolymer tails to DNA molecules prior to the construction of recombinants.
a phosphate from ATP to the 5’ ends of either DNA or RNA. It is a product of T4 bacteriophage. The enzymatic activity of PNK is utilized in two types of reactions: a. Forward reaction: PNK transfer the gamma phosphate from ATP to the 5’ end of a polynucleotide (DNA or RNA) b. Exchange reaction: The target DNA or RNA consisting of a 5’-PO is incubated with an excess of ADP. PNK transfers the phosphate from the nucleic acid onto an ADP and forms ATP and leaves a dephosphorylated target. Then PNK carries out a forward reaction and transfers a 5’-PO group from ATP onto the target nucleic acid.
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