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Genetics and Evolutionary Biology pt2 > The Polymerase Chain Reaction > Flashcards

The Polymerase Chain Reaction Flashcards

1
Q

When was PCR first created?

A
  • Until the mid-1980s, the only way to make many copies of DNA was to insert the DNA into E. coli.
  • In 1985, Kary Mullis invented a precise and radical new method of selecting and amplifying a section of DNA, the polymerase chain reaction (PCR).
  • In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.
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2
Q

PCR is now a common and often indispensable technique used in medical and biological research labs, what are some of the activities it is used for?

A
  • DNA cloning
  • DNA cloning for sequencing
  • DNA-based phylogeny
  • Functional analysis of genes
  • Diagnosis of hereditary diseases
  • Identification of genetic fingerprints (used in forensic sciences and paternity testing)
  • Detection and diagnosis of infectious diseases
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3
Q

What is the PCR?

A
  • Polymerase chain reaction is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
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4
Q

What does PCR do?

A
  • The Polymerase chain reaction uses repeated cycles of heating and cooling to make many copies of a specific region of DNA.
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5
Q

What is the first half of the first cycle of the polymerase chain reaction?

A
  • First the temperature is raised to near boiling (about 95 degrees), causing the double stranded DNA to separate or denature into two single strands.
  • When the temperature is decreased (about 55 degrees), short DNA sequences known as primers bind or anneal to complementary matches on the target DNA sequence
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6
Q

What is the second half of the first cycle of the polymerase chain reaction?

A
  • The primers (made by the scientists) bracket the target sequence to be copied.
  • At a slightly higher temperature (about 72 degrees), the enzyme tac ploymerase binds to the primed sequences and adds nucleotide’s to extend the second strand. This completes the first cycle
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7
Q

What happens after the first cycle of the polymerase chain reaction?

A
  • In subsequent cycles, the process of denaturing, annealing and extending are repeated to make additional DNA copies
  • After 3 cycles, the target sequences, defined by the primers begins to accumulate
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8
Q

How many target sequences can be made?

A
  • After 30 cycles, up to a billion copies of the target sequence are produced from a single starting molecule
  • To get to this amount from a single starting molecule can take as little as 2 hours
  • Up to 7h and about 25 - 35 cycles
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9
Q

What is DNA Amplification?

A
  • The production of multiple copies of a sequence of DNA. Repeated copying of a piece of DNA. A tumor cell amplifies, or copies, DNA segments as a result of cell signals and sometimes environmental events
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10
Q

What are Oligonucleotide primers?

A
  • Synthetic nucleotides (oligonucleotide primers) complementary to known flanking sequences are used to prime enzymatic amplification of the sequence of interest
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11
Q

What does the Polymerase Chain Reaction need to function?

A
  • Synthetic oligonucleotide primers complementary to known sequence
  • dNTPs (deoxynucleotide triphosphate, ie. dATP, dTTP, dGTP, dCTP)
  • Template DNA
  • Mg2+
  • DNA polymerase (eg. Taq polymerase)
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12
Q

What are the three repeated steps in the cycles of the PCR?

A
  • Denaturation of DNA (92 95*C)
  • Annealing of denatured DNA to oligonucleotide primers (50-60*C)
  • Replication of the DNA segment between the sites complementary to the primers (70-72*C) by DNA polymerase
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13
Q

How does amplification benefit the PCR?

A
  • Amplification occurs exponentially; each cycle doubles the number of molecules of the sequence of interest
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14
Q

Study the diagrams of the PCR - Cycle 1, PCR - Cycle 2 and the Repeat cycles of the PCR

A

https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing

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15
Q

What DNA is used for the starting material for the PCR?

A
  • A sample of chromosomal DNA or Genomic DNA can be used
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16
Q

Why is Taq polymerase used in the PCR? What are its limitations?

A
  • DNA polymerase from Thermus aquaticus is used for PCR because it is heat-stable
  • Taq polymerase lacks proofreading activity, so errors are introduced into the amplified DNA at low but significant frequencies: 1 - 2 x 10^-5 errors/bp
17
Q

Why is Pfu Polymerase used in the PCR?

A
  • When high fidelity (exactness) is required, heat-stable polymerases with proofreading activity are used instead of Taq (eg. Pfu polymerase from Pyrococcus furiosus): 3’-5’ exonuclease (proofreading) activity (1.6 x 10^-6 errors/bp)
  • Pfu polymerase is used for site-directed mutagenesis (come back to this later)
18
Q

What is the first major limitation of PCR?

A
  • Requires some prior knowledge of target DNA sequence in order to design/construct primers
19
Q

What is the 2nd major limitation of PCR?

A
  • Because PCR can amplify DNA present in extremely low amounts, contamination is a problem (ie. Amplification of non-target/contaminating DNA)
20
Q

What is the 3rd major limitation of PCR?

A
  • Taq polymerase lacks proofreading activity (so makes errors) and usually can only amplify up to 2000 bp (however through optimization can get up to 50 kbp
21
Q

Why is the PCR important for genetics?

A
  • It is a standard tool in all Labs working with DNA (clone/amplify DNA of interest from any source)
  • Environmental DNA, and isolation of DNA from ancient sources (eg. Mummified remains)
  • Used to mutated genes/DNA at specific sites
  • Modification of PCR is used to determine mRNA levels of genes of interest (eg. Changes in gene expression following drug treatment)
22
Q

What are some applications of the PCR that are used in society?

A
  • Diagnostic tests (eg. HIV and other viruses in blood, bacterial infections (eg. Chlamydia, Neisseria, Mycobacterium)
  • Identify genetic variation in natural populations (eg. Conservation biology, Forensic DNA fingerprinting)
23
Q

What is reverse transcription?

A
  • A reverse transcriptase is an enzyme used to generate complementary DNA from an RNA template, a process termed reverse transcription
24
Q

What is reverse transcription used in?

A
  • It is central to the infectious nature of retroviruses, several of which cause disease in humans, including human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), and human T-cell lymphotrophic virus I (HTLV-I), which causes leukemia.
  • Reverse transcriptase is also a fundamental component of a laboratory technology known as reverse transcription polymerase chain reaction (RT-PCR), a powerful tool used in research and in the diagnosis of diseases such as cancer.
25
Q

What is Reverse Transcriptase PCR?

A
  • Reverse transcription polymerase chain reaction is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction. It is primarily used to measure the amount of a specific RNA.
26
Q

How does reverse transcriptase work?

A
  • Eukaryotes can only express DNA not RNA
  • What a virus does is hijack the cellular mechanism (central dogma) and uses it to replicate itself
  • It needs just one enzyme to turn its own viral RNA into DNA, which can then be used by the cell its invading which can then be used to make more viruses.
  • Reverse transcriptase (the one enzyme needed) uses the viral RNA, turns it into DNA and it can then insert it’s DNA into your genome. Then as your chromosomes are replicating they’re also replicating the virus
27
Q

Study the diagrams for Reverse transcriptase.

A

https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing

28
Q

What is site-directed mutagenesis?

A
  • Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products.
29
Q

What can Site-directed Mutagenesis (using PCR) be used for?

A
  • This technique can be used to introduce many types of specific mutations into the gene of interest, for example:
    • Single nucleotide changes
    • Deletions
    • Insertions (small or large)
30
Q

What are the four different types of mutations that site-directed mutagenesis includes?

A
  • Point mutation
  • Deletion mutation
  • Large Insertion mutation
  • Small Insertion mutation
  • Remember that insertion and deletion mutations are both frameshift mutations
31
Q

What is the first two steps in the process of site-directed Mutagenesis (using PCR)?

A
  • Starts with double stranded, circular DNA known as a plasmid which has the gene of interest in it that has a target site/sequence
  • Next, the plasmid is denatured and primers with the desired mutation are annealed to either side of the point where you want the mutation to occur
32
Q

What are the last three steps in the process of site-directed Mutagenesis (using PCR)?

A
  • The PCR (using pfu polymerase) begins to amplify the mutation
  • Once the PCR has amplified the mutated gene, the DpnI restriction enzyme digests the methylated DNA
  • Lastly, the mutated plasmid undergoes ligation.
33
Q

What is the primer design for site-directed Mutagenesis (using PCR)?

A
  • Both primers must contain the desired mutation and anneal to the same sequence on opposite strands
  • Mutation should be in the middle of the primer with 10 15 bp of the correct sequence on either side
34
Q

Study the diagrams for Site-directed Mutagenesis (using PCR) and the process of site-directed Mutagenesis (using PCR)

A
  • Google doc
35
Q

Does the type of mutation affect the PCR during the site-directed mutagenesis?

A
  • Yes, the number of reaction cycles depends on the type of mutation, for example:
  • Point mutations have 12 cycles
  • Single amino acid changes have 16 cycles
  • Multiple amino acid deletions or insertions have 18 cycles
  • The table of these values is in the google doc

Genetics and Evolutionary Biology pt2 (7 decks)

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