Lec6 | PCR Flashcards
In what year was PCR developed?
PCR was developed in 1983.
Is PCR considered an in vivo or in vitro process?
PCR is considered an in vitro process.
What cellular process is PCR a laboratory version of?
PCR is a laboratory version of DNA Replication in cells.
What is a key capability of the PCR process regarding specific DNA sequences?
PCR can make billions of copies of a particular sequence of DNA in a short time.
What does the term in vivo signify in contrast to in vitro?
In vivo signifies occurring in a living cell, in contrast to in vitro occurring in a test tube.
What is one typical downstream use of PCR?
PCR is typically used to amplify DNA for downstream applications.
Does PCR amplify all of the DNA present in a sample?
PCR does not copy all the DNA in the sample.
PCR amplifies a very specific sequence of genetic code from template DNA.
What molecules target specific sequences in PCR?
PCR primers target specific sequences in PCR.
What information about the target sequence must be known in order for PCR to function correctly?
It does require the knowledge of some DNA sequence information which flanks the fragment of DNA to be amplified.
What does the application of PCR enable in terms of disease diagnosis?
PCR enables healthcare professionals to diagnose infections and initiate treatment more quickly.
How is PCR used in genetic testing?
PCR is used to detect mutations in specific genes in genetic testing.
What does the application of PCR enable in the field of drug development?
PCR helps to identify and validate drug targets and to screen potential drug candidates in drug development.
In the field of forensic analysis, what is a major use of PCR?
In forensic analysis, PCR is used to identify and match DNA samples from crime scenes.
What are the essential components of a PCR reaction?
A PCR reaction requires a DNA sample, primers, nucleotides, Taq polymerase, mix buffer, a PCR tube, and a thermal cycler.
Why is MgCl2 added to the PCR mixture?
MgCl2 enhances the enzymatic activity of DNA polymerase, increasing the efficiency of DNA amplification.
What piece of equipment precisely changes temperatures during PCR cycles?
The thermal cycler adjusts temperatures for denaturation, primer annealing, and extension in each PCR cycle.
What is the main purpose of the PCR cycle?
The PCR cycle repeatedly doubles the target DNA by denaturing strands, annealing primers, and extending new strands.
What is the first step in sample preparation for DNA extraction?
The first step is to prepare the sample by collecting and processing material before lysing cells for DNA isolation.
Why are red blood cells lysed during DNA extraction?
Red blood cells are lysed to remove hemoglobin and reduce contamination before releasing white blood cells for DNA retrieval.
What is the function of WBC lysis in DNA extraction?
WBC lysis breaks open white blood cells, releasing their DNA for subsequent binding and purification steps.
How is DNA retained during the DNA binding step?
DNA binds selectively to a column or matrix under specific conditions, allowing separation from impurities.
Why is a wash step necessary in DNA extraction?
The wash step removes residual proteins and contaminants, leaving only the bound DNA on the column or matrix.
What happens during the elution step of DNA extraction?
The elution step releases purified DNA from the column into a buffer solution for downstream applications.
How does the lysis process liberate DNA?
Lysis breaks cells and nuclear membranes to release DNA and dissolves proteins using mechanical or enzymatic methods (Proteinase K).
What does the precipitation step achieve in DNA extraction?
Precipitation separates DNA from cellular debris by adding sodium ions and ethanol, causing DNA to aggregate.
How is DNA further purified after precipitation?
DNA is repeatedly washed to remove contaminants and then eluted in a slightly alkaline buffer for stability.
How is the purity of extracted DNA commonly verified?
DNA purity is checked with a nanodrop or spectrophotometer, where an absorbance ratio near 1.8 suggests pure DNA.
An absorbance ratio above ~1.8-2 suggests pure RNA.
Why are two primers chemically synthesized based on DNA sequencing information?
Two primers are needed so each DNA strand has a complementary primer for efficient amplification at the 3’ end.
Why can DNA polymerase only add nucleotides to a primer’s 3’ end?
DNA polymerase requires a pre-existing 3’-OH group to attach new nucleotides and extend the DNA strand.
Why do we use two primers in a PCR reaction instead of one?
Two primers ensure both strands of the target DNA are amplified, rather than just a single strand.
How does a forward primer function in PCR?
The forward primer matches the forward DNA strand sequence and anneals to the complementary reverse strand.
What is the role of the reverse primer in PCR?
The reverse primer matches the reverse DNA strand sequence and anneals to the complementary forward strand.
Why should primers avoid runs of four or more identical bases?
Long runs of a single base can cause primer-dimer formation and reduce amplification specificity.
What melting temperature range is recommended for PCR primers?
PCR primers generally require melting temperatures between 55°C and 70°C for optimal annealing.
Why must two PCR primers have melting temperatures within 5°C of each other?
Closely matched melting temperatures ensure both primers anneal efficiently at the same temperature cycle.
What is the ideal GC content for PCR primers?
PCR primers should have a GC content between 40% and 60% to maintain stable and specific binding.
How long are PCR primers typically designed to be?
PCR primers are usually between 18 and 30 nucleotides in length for effective amplification.
What are the three distinct steps of a PCR cycle governed by temperature?
The PCR cycle is composed of denaturation, annealing, and extension steps.
At approximately what temperature does the denaturation step occur, and what is its purpose?
Denaturation typically occurs at around 95°C to separate double-stranded DNA into single strands.
Why is the PCR reaction rapidly cooled to a lower temperature after denaturation?
The reaction is cooled to about 50–65°C so primers can anneal to their complementary single-stranded DNA templates.
What temperature is commonly used for the extension step in PCR, and which enzyme works best there?
The extension step commonly occurs at 72°C, which is optimal for thermostable DNA polymerases like Taq.
How are double-stranded DNA molecules separated during denaturation?
High temperature (94–98°C) breaks the hydrogen bonds between base pairs, producing single-stranded DNA.
Why might the denaturation step take up to two minutes?
Denaturation can take up to two minutes because higher GC content requires increased temperature or time to fully separate the DNA strands.
What is the critical function of primer annealing during the annealing step?
Primers hybridize specifically to complementary target sequences, enabling selective amplification of the DNA region of interest.
How should the annealing temperature be set in relation to primer melting temperatures?
The annealing temperature should be about 5°C below the lowest melting temperature of the two primers.
What happens if the annealing temperature is too high or too low?
A temperature set too high prevents primer binding, whereas a temperature set too low causes non-specific primer attachment.
How long is the annealing step typically maintained during PCR?
The annealing step generally lasts between 15 and 60 seconds, with an optimal duration of about 30 seconds.
What key reaction occurs during the extension step of PCR?
The thermostable DNA polymerase synthesizes new DNA strands by adding nucleotides to the annealed primers, exponentially amplifying the target DNA.
In what temperature range and time frame does the PCR extension step usually occur?
Extension often takes place at 70–80°C for 1–2 minutes, depending on the polymerase and the size of the DNA fragment.
How does Taq polymerase extend DNA during PCR?
Taq polymerase operates optimally at 70–80°C, taking about one minute for the first two kilobases and adding one minute per additional kilobase.
Under what conditions does Pfu polymerase function during PCR extension?
Pfu polymerase typically extends DNA at 75°C, requiring around two minutes per kilobase for efficient replication.
