Chapter 20

What is a plasmid?

A small circular DNA molecule often used as a vector in gene cloning

This technique enables researchers to determine the DNA bases in genes and other chromosomal regions.

DNA sequencing

The DNase I footprinting technique is used to study _________ _________ interactions

Protein DNA

The ___________ probe is an oligonucleotide that can be used to follow real-time PCR. It has a reporter molecule at one end and a quencher molecule at the other end.

TaqMan

To perform many cycles of PCR, you need a machine that can change temperatures at exact times. What do you call this machine?

Thermocycler

You wish to determine if a protein is made at a particular stage of development. What technique would you use?

Western blotting

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

Match the phase of real-time PCR with the accumulation of products.

-Exponential- The amount of product nearly doubles with each cycle but may be difficult to detect as the amounts are small. -Linear- Accumulation of the product shows a directly proportional relationship to cycle number. -Plateau- Accumulation of the product levels off as one or more reagents are used up.

Place the steps in a real-time PCR experiment in order from first to last, putting the first step at the top.

1. A primer and the TaqMan probe both anneal to template DNA. 2. Taq polymerase cleaves the oligonucleotide in TaqMan 3. The report can emit unquenched fluorescence that can be measured 4. More and more TaqMan probes are digested and the level of fluorescence increases.

What activity of Taq polymerase separates the reporter from the quencher in the TaqMan molecule?

5' to 3' exonuclease

What is a DNA library?

A collection of recombinant vectors

What is a cloning vector?

A molecule that carries the DNA to be cloned

In gene cloning, what is the vector?

A small DNA molecule that can replicate independently within a host cell

How is the amount of DNA produced during real-time PCR measured?

By measuring the fluorescence emitted by the probe added to the PCR mixture

Which component in the CRISPR-Cas9 system makes a double-strand break in DNA?

Cas9

Dideoxy sequencing was formulated based on scientists' knowledge of what process?

DNA replication

Knowing the sequence of the DNA ______ the gene of interest allows scientists to design appropriate primers.

Flanking

In gene cloning, how is a suitable vector chosen?

It must replicate in the appropriate cell type

Why would you use a poly-dT primer when making cDNA?

It would be complementary to the poly-A tail at the 3' end of the mRNA.

Which scientist developed the polymerase chain reaction?

Kary Mullis

In PCR, the temperature must be ______ from the denaturation temperature in order for primers to anneal.

Lowered

Which scientist(s) developed an early method of DNA sequencing that involved base-specific chemical cleavage of DNA?

Maxam and Gilbert

How can PCR amplify one segment of DNA from a complex mixture of potential template molecules?

Primers can be designed to flank a specific segment of DNA.

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.

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

Why would one use a vector with a selectable marker?

To identify cells containing the vector

What is the purpose of gene cloning?

To produce many copies of a DNA molecule of interest

True or false: Amplifying a gene by PCR results in many copies, just like cloning using a vector and host cell.

True

True or false: PCR can amplify one segment of DNA from a mixture.

True

A small DNA molecule that can replicate independently within a host cell and thus make many copies of an inserted gene is called a___________

Vector

What technique is used to identify a particular protein in a mixture of proteins?

Western blotting

In PCR, each cycle uses the products of the previous cycle as templates. What do you call this?

a chain reaction

Site-directed mutagenesis allow a researcher to make a mutation ______.

at a specific sequence of DNA

Many species of bacterial cells make restriction enzymes to protect themselves from invasion by

bacteriophages

A particular gene to be cloned is often isolated from ______.

chromosomal DNA

If a gene is amplified by PCR so that there are many copies, it can be said to be ______.

cloned

Cells that can take up DNA from the medium are considered ________ cells

competent

Transformation occurs when ______.

competent cells take up DNA from the medium

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

The DNA sequencing method developed by Frederick Sanger that became a commonly used method of DNA sequencing is called ________ sequencing.

dideoxy

If the oxygens on carbons 2 and 3 of the sugar of a nucleotide have been removed, the nucleotide is referred to as a

dideoxyribonucleotide

The gel retardation assay is also known as the ______.

electrophoretic mobility shift assay

What technique is useful for studying protein-DNA interactions?

electrophoretic mobility shift assay

Antibodies bind to three-dimensional structures found within a protein that are called ______.

epitopes

When using PCR to amplify DNA, short oligonucleotides called primers ______.

flank the region of DNA to be amplified

A recircularized vector is one that has ______.

ligated with itself

A major advantage of CRISPR-Cas technology over site-directed mutagenesis is that it can be used directly on _______ cells

living

In gene cloning, cells are treated with agents that ______, creating competent cells.

make them permeable to DNA

A small circular DNA molecule that is often used as a vector in gene cloning is called a(n)

plasmid

In 1985, Kary Mullis developed a way to copy DNA without vectors or host cells. This technique is called

polymerase chain reaction

Short oligonucleotides that flank the region of DNA to be amplified by PCR are called

primers

A vector that contains a piece of chromosomal DNA is referred to as a __________ vector

recombinant

Enzymes that bind to a specific DNA sequence and cut the DNA backbone are called

restriction enzymes

The enzyme that uses RNA as a template to make a complementary strand of DNA is called

reverse transcriptase

The Maxam and Gilbert method of DNA sequencing used chemicals that cleaved the DNA at ______.

specific bases

In PCR, the template DNA is ______.

the DNA to be amplified

To perform PCR, a machine called a __________ automates the timing of each cycle.

thermocycler

The natural function of CRISPR-Cas system in bacteria is to _______

to provide defense against bacteriophages'

True or false: Chromosomal DNA is a common source of cloned DNA.

true

What type of apparatus does one need to quantify the DNA produced during real-time PCR?

A thermocycler that can detect fluorescence

How is the TaqMan probe used to monitor real-time PCR?

As amplification increases, fluorescence increases.

A DNA molecule that acts as a carrier of DNA that is to be cloned is called a(n) ______.

Cloning vector

In PCR, the two primers bind to specific sites in the __________ and flank the gene to be amplified.

DNA

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 library

DNA sequencing enables researchers to determine the order of ______ ______ in a gene.

DNA nucleotides

Which of the following techniques is used to study protein-DNA interactions?

DNase I footprinting

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

___________-__________ PCR allows one to assess the amount of DNA produced during a PCR amplification as it is happening.

Real time

What is the technique that allows one to determine the amount of template DNA present when the PCR cycles began?

Real-time PCR

_______________ DNA technology uses in vitro molecular techniques that combine DNA fragments to produce novel arrangements.

Recombinant

A resistance gene that allows a host cell containing a vector to grow on a toxic substance is called a(n) ____________

Selective markers

_________-_________ _________ allows a researcher to produce a mutation at a specific sequence.

Site Directed Mutagenesis

How does a bacterial cell use restriction enzymes?

To protect the cell against invasion by bacteriophages

You would_________ a gene to make many copies of that gene

clone

Restriction endonucleases are used in gene cloning to ______.

cut the DNA backbone prior to inserting the DNA to be cloned

Reverse transcriptase PCR can be used to ______.

detect and quantify the amount of a specific RNA.

An antigen has one or more three dimensional structures called ______ to which a(n) ______ will bind.

epitopes; antibody

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

In automated sequencing, each dideoxyribonucleotide is labeled with a different colored ______.

fluorescent dye

A cell that harbors a vector is called a _________

host cell

What is the term that describes a cell that contains a DNA cloning vector?

host cell

CRISPR-Cas technology has been used to mutate genes in ______.

human cell lines adult mice plant cells roundworms mouse embryos

Primers are chosen for PCR based on ______.

knowing the DNA sequence flanking the gene of interest

If the two ends of a vector cut with a restriction enzyme ligate back together without an insert, a _________ vector has been created.

recircularized

The process by which competent cells take up DNA from the extracellular medium is called

transformation

In dideoxyribonucleotides, __________ oxygens are removed from the sugar compared with ribose.

two

Order the steps in one cycle of a PCR reaction, putting the first step at the top.

1. Denaturation 2. Primer annealing 3. Primer extension

During PCR, the process of ______ _______

results when the Taq polymerase catalyzes the synthesis of complementary DNA, starting at the primers.

Primer annealing occurs when ______.

short oligonucleotides bind to complementary DNA flanking the gene of interest

The enzyme ______ ________ is used when PCR is employed to detect and quantify the amount of a specific RNA.

reverse transcriptase

A resistance gene that allows a host cell containing a vector to grow on a toxic substance is called a(n)

selectable marker

The use of dideoxyribonucleotides with different colored fluorescent dyes allows the detection of the ______.

sequence of DNA