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