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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Published on: May 22, 2012
The expanding polymerase universe
1University of Southern California, Department of Biological Sciences and Chemistry, Stauffer Hall of Science 172, Los Angeles, California 90089-1340, USA. mgoodman@rcf.usc.edu
Nature Reviews. Molecular Cell Biology
|March 20, 2001
Summary
Newly discovered DNA polymerases copy damaged DNA bases. Researchers are now investigating their roles in DNA repair, mutation, and cancer avoidance.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The number of known prokaryotic and eukaryotic DNA polymerases has significantly increased recently.
- Many newly identified DNA polymerases possess the ability to replicate aberrant bases within DNA templates.
- These enzymes handle DNA structures that more conventional polymerases avoid.
Purpose of the Study:
- To elucidate the diverse functions of recently discovered DNA polymerases.
- To understand the roles of these enzymes in various cellular processes, including DNA repair and mutation.
Main Methods:
- Functional characterization of novel DNA polymerases.
- Assays to determine substrate specificity and processivity.
- In vivo and in vitro studies to assess roles in DNA lesion bypass and repair.
Main Results:
- Identified novel DNA polymerases capable of copying damaged DNA bases.
- Evidence suggests these enzymes are involved in error-prone DNA synthesis.
- Preliminary data indicate roles in processes like somatic hypermutation and DNA double-strand break repair.
Conclusions:
- The expanding family of DNA polymerases plays critical roles in managing DNA damage and genomic integrity.
- Further research is essential to fully understand their implications in processes ranging from cancer to replication fidelity.
Related Concept Videos
01:32PCR
Overview
03:01The Replisome
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
00:43Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
00:43Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
03:01The Replisome
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
01:31Proofreading
Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore, it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Errors During Replication are Corrected by the DNA Polymerase Enzyme