Nucleic acid enzymes Flashcards
purpose of nucleic acid enzymes
- Synthesize longer polymers
- Degrade nucleic acid into shorter fragments
• Critical for DNA replication and transcription and must be present in all replicating cells
emzyme use in diagnostics
Enzymes are used extensively in nucleic acid diagnostics for: • Sample preparation • Probe labeling • Signal generation • Amplification
unique enzymes
Unique enzymes, found in bacteria and viruses, act on specific nucleic acid sequences
• Many of these have been purified and synthesized in vitro
• Some have been engineered with alterations that improve their performance or stability
ex, taq polymerase is from bacteroa & can withstand thermal changes
Ligase
catalyzes phosphodiester bonds to form between 2 nucleic acid chains
- seals up all the gaps in the DNA molecule, like the Okazaki fragments
DNA ligase is not sequence specific & requires the presence of complementary template
RNA ligase does not require a template but is sensitive to sequence
Nuclease
Enzymes that hydrolyze one or more phosphodiester bonds in nucleic
acid polymers
Exonucleases
• Cleave nucleotides one at a time from the ends
Endonucleases
• Act only on internal bonds
very specific; either DNA or RNA specific and may act on only double orsingle stranded polymers
• For example: DNASE I digests only double-stranded DNA; S1
nuclease acts only on single-stranded DNA
• DNASE I has been used to specifically degrade DNA in nucleic
acid mixtures when only RNA is of interest
RNAses are very stable enzymes that are difficult to remove – they
are common laboratory contaminants
- may require an extra level of QC to ensure RNA are intact
Restriction Endonuclease
Found in bacteria and degrade foreign DNA
sequence specific
At the location where this sequence is found, the
enzyme cuts both strands in a reproducible manner,
resulting in either staggered or blunt-end cuts
used for:
• Digesting large fragments of DNA into smaller pieces
• Preparing DNA from different sources to be joined together in cloning procedures
Nicking enzymes
restriction enzymes that cut only one strand of double-stranded nucleic acid
Methylation-sensitive enzymes
restriction enzymes that distinguish between cytosine and 5-methylcytosine
• In humans, methylation of cytosine is a common epigenetic modification of DNA which affects gene expression
Polymerase
catalyze synthesis of complementary nucleic acid polymers using a parent strand as a template
In vitro, these enzymes extend an oligonucleotide primer that is annealed to a template strand
- Extension requires that the 3’OH of the extending end is free and that the nucleotide triphosphates (NTP) are
present
- Extension ends when you run out of template or NTPs or if no 3’OH groups are available at the extending end
Thermostable polymerases are essential reagents
- stable at high temps
ex. thermus aquaticus ( taq) DNA polymerase
polymerase with 3’ to 5’ exonuclease activity
polymerase with 3’ to 5’ exonuclease activity is able to correct mismatched base pairs at the 3’ end of the
extending chain
• This proofreading activity increases polymerase fidelity by decreasing the number of mis-incorporated bases
- increases accuracy
polymerase with 5’ to 3’ exonuclease activity
increase the process ability of a polymerase by cleaving any
blocking probes or secondary structures
- can remove anything blocking it ex. miRNA
Reverse Transcriptase
Found in retroviruses
• Retroviruses have RNA genomes and reverse
transcriptase activity is required as part of their
replication
Catalyzes the synthesis of DNA from either an RNA or DNA template
• In vitro, reverse transcriptase is used to make
complementary DNA (cDNA) copies of RNA and may be
used for:
• Cloning
• Probe preparation
• Nucleic acid assays
- PCR & other amplification techniques can only amplify DNA genomes so if we have RNA genome virus we can make DNA to test it
