BIOL 3416: Chapter 20
What are the steps in cloning a gene?
1. Chromosomal DNA is isolated and cut with a restriction enzyme; the plasmid DNA is cut with the same enzyme 2. The digested chromosomal DNA and plasmid DNA are incubated together. 3. Ligation by DNA ligase
In PCR, each cycle uses the products of the previous cycle as templates. What do you call this?
A chain reaction
What is a DNA library?
A collection of recombinant vectors
In gene cloning, what is the vector?
A small DNA molecule that can replicate independently within a host cell
What is a plasmid?
A small circular DNA molecule often used as a vector in gene cloning
What type of apparatus does one need to determine the amount of DNA produced during quantitative PCR?
A thermocycler that can detect fluorescence
Plateau
Accumulation of the product levels off as one or more reagents are used up
Linear
Accumulation of the product shows a directly proportional relationship to cycle number
How is the amount of DNA produced during quantitative PCR measured?
By measuring the fluorescence emitted by the probe added to the PCR mixture
A researcher may use restriction enzymes to digest the DNA of an organism. The fragments of DNA are then ligated individually into many vectors. This collection of recombinant vectors is called a
DNA (or genomic) library
A technique used to determine the order of the nucleotides in DNA is called
DNA Sequencing
Dideoxy sequencing was formulated based on scientists' knowledge of what process?
DNA replication
This technique enables researchers to determine the DNA bases in genes and other chromosomal regions.
DNA sequencing
What are the steps in of one cycle of PCR
Denaturation, Primer annealing, Primer extension
In a PCR cycle, the first step is to treat the template DNA with heat, causing the strands to separate, a process called
Denature/ Denaturation
The DNA sequencing method developed by Frederick Sanger that became a commonly used method of DNA sequencing is called
Dideoxy Sequencing
To determine if PCR is successful, a researcher typically runs a sample of DNA on a gel, stains the DNA with a dye called _____ _____ (EtBr), and then observes the gel under UV light, which causes the dye to fluoresce.
Ethidium Bromide
How is ethidium bromide used to analyze amplification by PCR?
Ethidium bromide stains DNA, allowing the size of a fragment in a gel to be determined.
As long as all of the fragments have been generated using the same restriction enzyme, researchers can control the order in which three or more DNA fragments will connect to each other. True or False
False
What is the term that describes a cell that contains a DNA cloning vector?
Host cell
In gene cloning, how is a suitable vector chosen?
It must replicate in the appropriate cell type.
Which scientist developed the polymerase chain reaction?
Kary Mullis
This technique is used to identify a specific RNA molecule within a mixture of RNA molecules.
Northern blotting
A vector must contain the _____ that is recognized by the species of the host cell and allows the host cell to make lots of copies of the vector.
Origin of Replication
What do you call the DNA sequence in a vector that allows the replication enzymes of the cell to make lots of copies of the vector?
Origin of replication
In 1985, Kary Mullis developed a way to copy DNA without vectors or host cells. This technique is called
Polymerase Chain Reaction (PCR)
During PCR, the process of _____ _____ results when the Taq polymerase catalyzes the synthesis of complementary DNA, starting at the primers.
Primer Extension
Short oligonucleotides that flank the region of DNA to be amplified by PCR are called
Primers
The amount of PCR product made during a quantitative PCR reaction (qPCR or RT-qPCR) is detected by changes in the level of fluorescence emitted from ____ that are added to the PCR mixture.
Probe(s)
PCR is called a chain reaction because the newly made DNA strands, or _____, of each previous reaction are used as _____ in subsequent reactions
Product(s); Reactant(s) or Template(s)
What is the technique that allows one to determine the amount of template DNA present when the PCR cycles began?
Quantitative PCR
The method that allows researchers to assess the amount of DNA produced during a PCR amplification as it is happening is called
Quantitative PCR (qPCR)
The enzyme that uses RNA as a template to make a complementary strand of DNA is called
Reverse Transcriptase
You have a piece of RNA, and you want to synthesize a complementary strand of DNA. Which enzyme would you use?
Reverse transcriptase
Exponential
The amount of product nearly doubles with each cycle but may be difficult to detect as the amounts are small.
What is recombinant DNA technology?
The production of new arrangements of DNA
When cloning a gene, why must the chromosomal DNA and the plasmid DNA be cut with the same restriction enzyme?
The sticky ends of the plasmid DNA will be complementary to the sticky ends of the chromosomal DNA.
To perform PCR, a machine called a(n)_____ automates the timing of each cycle
Thermocycler
To perform many cycles of PCR, you need a machine that can change temperatures at exact times. What do you call this machine?
Thermocycler
In PCR, why do the primers bind to specific sites in the DNA on either side of the gene of interest?
They are complementary to the flanking sequences.
What is the purpose of Northern blotting?
To identify a specific RNA molecule within a mixture of RNA molecules
What is the purpose of gene cloning?
To produce many copies of a DNA molecule of interest
Chromosomal DNA is a common source of cloned DNA. True or False?
True
What technique is used to identify a particular protein in a mixture of proteins?
Western blotting
You wish to determine if a protein is made at a particular stage of development. What technique would you use?
Western blotting
Which of the following are limitations of using restriction enzymes for the construction of recombinant DNA molecules?
When connecting three or more DNA fragments, it is difficult to control the order in which those fragments will be connected to each other. The researcher cannot control the directionality of the hydrogen-bonding process.
The purpose of vector DNA is to
act as a carrier of a DNA sequence that is to be cloned
A particular gene to be cloned is often isolated from
chromosomal DNA
To make many copies of a gene, you would ____ that gene
clone or amplify
In PCR, primer extension refers to the synthesis of ______ starting at the primers.
complementary DNA
A recombinant vector
contains a piece of chromosomal DNA
A vector requires an origin of replication so that it can be____
copied many times by the host cell
Restriction endonucleases are used in gene cloning to _____
cut the DNA backbone prior to inserting the DNA to be cloned
The steps involved in PCR are, in order, _____, primer ______, and primer ______
denaturation (denature); annealing (anneal); Extension
Reverse transcriptase PCR can be used to
detect and quantify the amount of a specific RNA.
During the initial phase of a real-time PCR experiment, called the ______ phase, the amount of PCR products is small and reagents are not limiting, so the amount of product nearly doubles with each cycle.
exponential
When using PCR to amplify DNA, short oligonucleotides called primers
flank the region of DNA to be amplified
In automated sequencing, each dideoxyribonucleotide is labeled with a different-colored
fluorescent dye
During a PCR cycle, the strands of the template DNA is denatured by ______, which separates the strands.
heat
A cell that harbors a vector is called a(n)
host cell
In PCR, the temperature must be ______ from the denaturation temperature in order for primers to anneal.
lowered
A technique used to measure gene expression is RT-qPCR, which can determine how much _____ from a specific gene was in the original sample.
mRNA
In the technique called RT-qPCR, the starting material is
mRNA that has been reverse transcribed into DNA
A small circular DNA molecule that is often used as a vector in gene cloning is called a(n)
plasmid or plasmids
The polymerase chain reaction (PCR) produces many copies of the region between two _____ that bind to sequences flanking the gene of interest.
primer
The use of in vitro molecular techniques that combine DNA fragments to produce novel arrangements is called
recombinant DNA technology
copied many times by the host cell
recombinant vector
Enzymes that bind to a specific DNA sequence and cut the DNA backbone are called
restriction enzymes (or endonucleases)
The use of dideoxyribonucleotides with different-colored fluorescent dyes allows the detection of the _____
sequence of DNA
Primer annealing occurs when
short oligonucleotides bind to complementary DNA flanking the gene of interest
In PCR, the DNA to be amplified is called the
template DNA
In PCR, the template DNA is
the DNA to be amplified
To perform PCR, a machine called a(n) _____ automates the timing of each cycle.
thermocycler
A DNA molecule that acts as a carrier of DNA that is to be cloned is called a(n)
vector
A small DNA molecule that can replicate independently within a host cell and thus make many copies of an inserted gene is called a(n)
vector or plasmid