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Questions and Answers
What is the third step in nucleic acid extraction after solid-phase extraction?
What is the third step in nucleic acid extraction after solid-phase extraction?
- Denaturation of nucleic acid
- Solid-phase extraction
- Removal of contaminants and recovery of nucleic acid (correct)
- Addition of primers for PCR
What may be present in the aqueous solution after liquid-phase extraction of nucleic acid?
What may be present in the aqueous solution after liquid-phase extraction of nucleic acid?
- Water only
- Amino acids
- Ethanol only
- Residual salts and organic solvents (correct)
Which process is used to remove contaminants from nucleic acid in the third step of extraction?
Which process is used to remove contaminants from nucleic acid in the third step of extraction?
- Centrifugation
- Chromatography
- Alcohol precipitation (correct)
- Electrophoresis
What is the final goal of removing contaminants in nucleic acid extraction?
What is the final goal of removing contaminants in nucleic acid extraction?
Which type of extraction method involves recovery of nucleic acid from liquid phase?
Which type of extraction method involves recovery of nucleic acid from liquid phase?
What is used in the third step to separate nucleic acid from contaminants?
What is used in the third step to separate nucleic acid from contaminants?
What is the range for the optimal GC% content in primers?
What is the range for the optimal GC% content in primers?
What is the maximum recommended number of G's or C's at the 3' end of primers?
What is the maximum recommended number of G's or C's at the 3' end of primers?
What temperature range generally produces the best results for melting temperatures (Tm) of primers?
What temperature range generally produces the best results for melting temperatures (Tm) of primers?
What type of sequences should primer design avoid to prevent mispriming?
What type of sequences should primer design avoid to prevent mispriming?
What is the accepted optimal length range for primers?
What is the accepted optimal length range for primers?
What is the characteristic of the melting temperature (Tm) that produces the best PCR results?
What is the characteristic of the melting temperature (Tm) that produces the best PCR results?
Which factor is crucial to avoid in the primer sequence design to prevent mispriming?
Which factor is crucial to avoid in the primer sequence design to prevent mispriming?
What should not be present in excess within the last five bases from the 3' end of primers?
What should not be present in excess within the last five bases from the 3' end of primers?
'Runs' misprime when they are part of which type of sequence pattern?
'Runs' misprime when they are part of which type of sequence pattern?
'Mismatch at 3’end' should be avoided in primer design to prevent:
'Mismatch at 3’end' should be avoided in primer design to prevent:
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Study Notes
Nucleic Acid Extraction Methods
- Cell Lysis: breaking open cells to release nucleic acid into solution
- Methods: chemical, enzymatic, or physical
- Considerations: complete lysis, avoiding nucleic acid degradation, and performing lysis quickly
- Separation of DNA from Cellular Debris and Other Cellular Components
- Methods: liquid-phase extraction or solid-phase extraction
- Liquid-phase extraction: organic extraction and inorganic extraction
- Solid-phase extraction: column-based extraction and magnetic bead-based extraction
- Removal of Contaminants and Recovery of Nucleic Acid
- Using alcohol precipitation to remove contaminants and recover nucleic acid
- Obtaining pure nucleic acid for downstream analysis
Polymerase Chain Reaction (PCR)
- Definition: amplification of a specific section of DNA from a DNA template
- Essential Components:
- Primers: short DNA fragments that bind to the target DNA region
- Nucleotides: building blocks for PCR product synthesis
- Reaction buffer: provides Mg2+ for DNA polymerase
- Steps:
- Denaturation (95°C): separating double-stranded DNA
- Annealing (50-65°C): primers bind to single-stranded template
- Extension (72°C): DNA polymerase synthesizes new DNA strand
- Cycle: repeated 20-30 times to amplify the target sequence
- Importance of primer design and specificity
- Applications: genotyping, RT-PCR, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing
- Optimization: primer design guidelines and PCR optimization
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