Lec6 | PCR

Question 1

In what year was PCR developed?

Answer 1

PCR was developed in 1983.

Question 2

Is PCR considered an in vivo or in vitro process?

Answer 2

PCR is considered an in vitro process.

Question 3

What cellular process is PCR a laboratory version of?

Answer 3

PCR is a laboratory version of DNA Replication in cells.

Question 4

What is a key capability of the PCR process regarding specific DNA sequences?

Answer 4

PCR can make billions of copies of a particular sequence of DNA in a short time.

Question 5

What does the term in vivo signify in contrast to in vitro?

Answer 5

In vivo signifies occurring in a living cell, in contrast to in vitro occurring in a test tube.

Question 6

What is one typical downstream use of PCR?

Answer 6

PCR is typically used to amplify DNA for downstream applications.

Question 7

Does PCR amplify all of the DNA present in a sample?

Answer 7

PCR does not copy all the DNA in the sample.

PCR amplifies a very specific sequence of genetic code from template DNA.

Question 8

What molecules target specific sequences in PCR?

Answer 8

PCR primers target specific sequences in PCR.

Question 9

What information about the target sequence must be known in order for PCR to function correctly?

Answer 9

It does require the knowledge of some DNA sequence information which flanks the fragment of DNA to be amplified.

Question 10

What does the application of PCR enable in terms of disease diagnosis?

Answer 10

PCR enables healthcare professionals to diagnose infections and initiate treatment more quickly.

Question 11

How is PCR used in genetic testing?

Answer 11

PCR is used to detect mutations in specific genes in genetic testing.

Question 12

What does the application of PCR enable in the field of drug development?

Answer 12

PCR helps to identify and validate drug targets and to screen potential drug candidates in drug development.

Question 13

In the field of forensic analysis, what is a major use of PCR?

Answer 13

In forensic analysis, PCR is used to identify and match DNA samples from crime scenes.

Question 14

What are the essential components of a PCR reaction?

Answer 14

A PCR reaction requires a DNA sample, primers, nucleotides, Taq polymerase, mix buffer, a PCR tube, and a thermal cycler.

Question 15

Why is MgCl2 added to the PCR mixture?

Answer 15

MgCl2 enhances the enzymatic activity of DNA polymerase, increasing the efficiency of DNA amplification.

Question 16

What piece of equipment precisely changes temperatures during PCR cycles?

Answer 16

The thermal cycler adjusts temperatures for denaturation, primer annealing, and extension in each PCR cycle.

Question 17

What is the main purpose of the PCR cycle?

Answer 17

The PCR cycle repeatedly doubles the target DNA by denaturing strands, annealing primers, and extending new strands.

Question 18

What is the first step in sample preparation for DNA extraction?

Answer 18

The first step is to prepare the sample by collecting and processing material before lysing cells for DNA isolation.

Question 19

Why are red blood cells lysed during DNA extraction?

Answer 19

Red blood cells are lysed to remove hemoglobin and reduce contamination before releasing white blood cells for DNA retrieval.

Question 20

What is the function of WBC lysis in DNA extraction?

Answer 20

WBC lysis breaks open white blood cells, releasing their DNA for subsequent binding and purification steps.

Question 21

How is DNA retained during the DNA binding step?

Answer 21

DNA binds selectively to a column or matrix under specific conditions, allowing separation from impurities.

Question 22

Why is a wash step necessary in DNA extraction?

Answer 22

The wash step removes residual proteins and contaminants, leaving only the bound DNA on the column or matrix.

Question 23

What happens during the elution step of DNA extraction?

Answer 23

The elution step releases purified DNA from the column into a buffer solution for downstream applications.

Question 24

How does the lysis process liberate DNA?

Answer 24

Lysis breaks cells and nuclear membranes to release DNA and dissolves proteins using mechanical or enzymatic methods (Proteinase K).

Question 25

What does the precipitation step achieve in DNA extraction?

Answer 25

Precipitation separates DNA from cellular debris by adding sodium ions and ethanol, causing DNA to aggregate.

Question 26

How is DNA further purified after precipitation?

Answer 26

DNA is repeatedly washed to remove contaminants and then eluted in a slightly alkaline buffer for stability.

Question 27

How is the purity of extracted DNA commonly verified?

Answer 27

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.

Question 28

Why are two primers chemically synthesized based on DNA sequencing information?

Answer 28

Two primers are needed so each DNA strand has a complementary primer for efficient amplification at the 3’ end.

Question 29

Why can DNA polymerase only add nucleotides to a primer’s 3’ end?

Answer 29

DNA polymerase requires a pre-existing 3’-OH group to attach new nucleotides and extend the DNA strand.

Question 30

Why do we use two primers in a PCR reaction instead of one?

Answer 30

Two primers ensure both strands of the target DNA are amplified, rather than just a single strand.

Question 31

How does a forward primer function in PCR?

Answer 31

The forward primer matches the forward DNA strand sequence and anneals to the complementary reverse strand.

Question 32

What is the role of the reverse primer in PCR?

Answer 32

The reverse primer matches the reverse DNA strand sequence and anneals to the complementary forward strand.

Question 33

Why should primers avoid runs of four or more identical bases?

Answer 33

Long runs of a single base can cause primer-dimer formation and reduce amplification specificity.

Question 34

What melting temperature range is recommended for PCR primers?

Answer 34

PCR primers generally require melting temperatures between 55°C and 70°C for optimal annealing.

Question 35

Why must two PCR primers have melting temperatures within 5°C of each other?

Answer 35

Closely matched melting temperatures ensure both primers anneal efficiently at the same temperature cycle.

Question 36

What is the ideal GC content for PCR primers?

Answer 36

PCR primers should have a GC content between 40% and 60% to maintain stable and specific binding.

Question 37

How long are PCR primers typically designed to be?

Answer 37

PCR primers are usually between 18 and 30 nucleotides in length for effective amplification.

Question 38

What are the three distinct steps of a PCR cycle governed by temperature?

Answer 38

The PCR cycle is composed of denaturation, annealing, and extension steps.

Question 39

At approximately what temperature does the denaturation step occur, and what is its purpose?

Answer 39

Denaturation typically occurs at around 95°C to separate double-stranded DNA into single strands.

Question 40

Why is the PCR reaction rapidly cooled to a lower temperature after denaturation?

Answer 40

The reaction is cooled to about 50–65°C so primers can anneal to their complementary single-stranded DNA templates.

Question 41

What temperature is commonly used for the extension step in PCR, and which enzyme works best there?

Answer 41

The extension step commonly occurs at 72°C, which is optimal for thermostable DNA polymerases like Taq.

Question 42

How are double-stranded DNA molecules separated during denaturation?

Answer 42

High temperature (94–98°C) breaks the hydrogen bonds between base pairs, producing single-stranded DNA.

Question 43

Why might the denaturation step take up to two minutes?

Answer 43

Denaturation can take up to two minutes because higher GC content requires increased temperature or time to fully separate the DNA strands.

Question 44

What is the critical function of primer annealing during the annealing step?

Answer 44

Primers hybridize specifically to complementary target sequences, enabling selective amplification of the DNA region of interest.

Question 45

How should the annealing temperature be set in relation to primer melting temperatures?

Answer 45

The annealing temperature should be about 5°C below the lowest melting temperature of the two primers.

Question 46

What happens if the annealing temperature is too high or too low?

Answer 46

A temperature set too high prevents primer binding, whereas a temperature set too low causes non-specific primer attachment.

Question 47

How long is the annealing step typically maintained during PCR?

Answer 47

The annealing step generally lasts between 15 and 60 seconds, with an optimal duration of about 30 seconds.

Question 48

What key reaction occurs during the extension step of PCR?

Answer 48

The thermostable DNA polymerase synthesizes new DNA strands by adding nucleotides to the annealed primers, exponentially amplifying the target DNA.

Question 49

In what temperature range and time frame does the PCR extension step usually occur?

Answer 49

Extension often takes place at 70–80°C for 1–2 minutes, depending on the polymerase and the size of the DNA fragment.

Question 50

How does Taq polymerase extend DNA during PCR?

Answer 50

Taq polymerase operates optimally at 70–80°C, taking about one minute for the first two kilobases and adding one minute per additional kilobase.

Question 51

Under what conditions does Pfu polymerase function during PCR extension?

Answer 51

Pfu polymerase typically extends DNA at 75°C, requiring around two minutes per kilobase for efficient replication.