BIO282 Lecture Notes - Lecture 4: Klenow Fragment, Alkaline Phosphatase, Exonuclease
Nucleic Acids Manipulation and Detection Methods
Lecture Outline:
• DNA manipulating enzymes
a. Nucleases-
i. Exonucleases
ii. Endonucleases
i. Restriction enzymes
i. Nomenclature and
ii. Cleavage sites
b. Ligases
c. Modifying enzymes
i. Alkaline phosphatase
ii. Polynucleotide kinase
iii. Terminal deoxynucleotide transferase
iv. Klenow fragment
DNA manipulating enzymes
Molecular techniques utilize a number of enzymes to manipulate DNA including
• Nucleases
• Ligases
• Modifying enzymes
-eases means it cuts it suffix (i.e. nucleases - cuts nucleic acids)
• Nucleases
• Nucleases cut DNA by breaking the phosphodiester bonds (not the hydrogen bonds
between chains) that link nucleotides in a DNA strand.
• Exonucleases:
• Endonucleases:
• Exonucleases: remove nucleotides one by one from the end of DNA
Example-1
Bal31-removes nucleotides from both chains of a double stranded DNA
• Size of the end product DNA will depend on the incubation time with the enzyme
Example-2
Exonuclease III- degrades DNA from 3` end but 3` overhangs are resistant to the
enzyme.
• It can be used to generate single stranded DNA
• It can be used to generate ‘Nested deletions’ from one end
o Reduced the length of overhang from one end only
o You can now determine where the promotor genes in DNA are
o Endonucleases: break internal bonds within DNA
Examples
i. Single strand specific enzyme
S1 nuclease: only cleaves single strands
Usually used to trim DNA molecules (if there are overhangs)
ii. Non-specific enzyme
DNase I: cuts both single and double stranded DNA. It cuts non-
specifically leaving mononucleotides and short oligonucleotides as end
product.
It will cut all the DNA except the part protected by protein (so you can
determine where protein binding occurs)
iii. Restriction enzymes cut double stranded DNA by recognizing specific sequences
Doesn’t start at the ends
Only when they recognize a specific sequence will they cut
• Microorganisms make restriction enzymes (also known as restriction endonucleases) to
protect themselves from foreign DNA.
Restriction Enzymes.
They restrict entry into the DNA but we use it in a different way. We use it to cut
nucleic acids.
Exo means start cutting from the ends and the ENDO means you start cutting
somewhere inside the DNA molecule.
a. Type II restriction endonucleases
Cleave double stranded DNA at specific sites known as the recognition site. A
specific sequence of bases in the DNA
Restriction enzymes have been classified into four groups Type 1-Type 4. Here we will focus on
Type II enzymes
Document Summary
Molecular techniques utilize a number of enzymes to manipulate dna including: nucleases, modifying enzymes. Bal31-removes nucleotides from both chains of a double stranded dna. Size of the end product dna will depend on the incubation time with the enzyme. Exonuclease iii- degrades dna from 3 overhangs are resistant to the enzyme. It can be used to generate single stranded dna. It can be used to generate nested deletions" from one end: reduced the length of overhang from one end only, you can now determine where the promotor genes in dna are, endonucleases: break internal bonds within dna. Usually used to trim dna molecules (if there are overhangs) Dnase i: cuts both single and double stranded dna. It cuts non- specifically leaving mononucleotides and short oligonucleotides as end product. It will cut all the dna except the part protected by protein (so you can determine where protein binding occurs)
