Intro to Molecular Bio Connect 20.1

Place the steps in a quantitative 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 reporter can emit unquenched fluorescence that can be measured 4. More and more TaqMan probes are digested and the level of fluorescence increases

Order the following steps in cloning a gene, putting the first step at the top.

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

Chain termination occurs when a dideoxyribonucleotide is incorporated into a growing DNA strand because there is no ______.

3' OH group

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

5' to 3' exonuclease

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

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

As amplification increases, fluorescence increases.

Select all vectors that can be used to clone large segments of DNA.

BACs YACs Cosmids

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

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

DNA nucleotides

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

DNA replication

Primers are chosen for PCR based on knowing the ______

DNA sequences flanking the gene of interest

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

DNA sequencing

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

Denaturation Primer annealing Primer extension

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.

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

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

Quantitative PCR

You have a piece of RNA, and you want to synthesize a complementary strand of DNA. Which enzyme would you use?

Reverse transcriptase

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

Why is Taq polymerase used in PCR?

The DNA polymerase must be thermostable as PCR involves cycles of heating.

Following exposure to a plasmid containing the ampicillin resistance gene, a bacterial cell that was previously sensitive now grows in the presence of the antibiotic. What happened?

The bacterial cell was transformed with a plasmid carrying the ampicillin resistance gene.

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.

Taq polymerase was first isolated from a bacterium called ____ _____.

Thermus aquaticus

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 gene cloning?

To produce many copies of a DNA molecule of interest

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

a chain reaction

"Sticky ends" created b cutting DNA with a restriction enzyme are useful in cloning because they ______

are areas where 2 pieces of DNA can hydrogen bond

In dideoxy sequencing, if a dideoxyribonucleotide is incorporated into the growing strand of DNA, the strand can no longer grow as there is no 3' OH group. This is called ______.

chain termination

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

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

cloned

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

cloning vector

Cells that can take up DNA from the medium are considered to be _______cells.

competent

Transformation occurs when ______.

competent cells take up DNA from the medium

DNA made using RNA as the starting material is called ______ DNA.

complementary

When DNA is made using RNA as the starting material, the DNA is called ______ DNA.

complementary

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

A method in which the exponential phase of real-time PCR is analyzed to quantitate the initial template concentration is called the ____ ____ method.

cycle threshold

Select the reagents needed to make cDNA.

dNTPs mRNA Poly-dT primer Reverse transcriptase

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

ddNTP (dideoxribonucleotide)

Reverse transcriptase PCR can be used to ______.

detect and quantify the amount of a specific RNA.

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

dideoxy

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

Viruses cannot be used as vectors in gene cloning

false

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

flank the region of DNA to be amplified

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

flanking

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

genomic/DNA library

If bacteria are transformed with a plasmid carrying an antibiotic-resistance gene, one would expect progeny of that cell to ______ when exposed to the antibiotic.

grow

A cell that harbors a vector is called a(n) _________ cell.

host

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

host cell

When cloning a gene into a vector, the sugar-phosphate backbone of each DNA molecule is covalently linked by the enzyme DNA

ligase

A recircularized vector is one that has

ligated with itself

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

lowered

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

make them permeable to DNA

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

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 ___ ___ ___(PCR).

polymerase chain reaction

During PCR, the process of _____ _____ results when the Taq polymerase catalyzes the synthesis of complementary DNA, starting at the primers.

primer extension

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

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

real time

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

recircularized

A vector that contains a piece of chromosomal DNA is referred to as a(n) ____ vector.

recombinant

The use of in vitro molecular techniques that combine DNA fragments to produce novel arrangements is called ________ DNA technology .

recombinant

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

restriction enzymes/endonucleases

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

selectable marker

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

selective marker

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

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

specific bases

Single-stranded stretches of DNA created by restriction enzymes, such as those shown on the left and right sides of the molecule in the figure, are called _____ ______.

sticky ends

In PCR, the DNA to be amplified is called the _______ DNA.

template

In PCR, the template DNA is _____

the DNA to be amplified

In real-time PCR, the cycle threshold is reached when ______

the accumulation of fluorescence is significantly greater than the background level

What is recombinant DNA technology?

the production of new arrangements of 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

Why would one use a vector with a selectable marker?

to identify cells containing the vector

How does a bacterial cell use restriction enzymes?

to protect the cell against invasion by bacteriophages

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

true

Chromosomal DNA is a common source of cloned DNA.

true

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(n)

vector or plasmid

Viruses can be used as ________ to carry other pieces of DNA.

vectors