CH 24 genetics
A molecule that has covalently linked DNA fragments from at least two sources is called a
Recombinant DNA molecule
What is the purpose of Southern blotting?
To identify a particular DNA sequence within a mixture of DNA fragments
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
How does a bacterial cell use restriction enzymes?
To protect the cell against invasion by bacteriophages
A vector requires an origin of replication so that it can be ______.
copied many times ~~~
common uses of gene cloning
* The expression of a cloned gene can be used to discover its cellular function. * Cloned genes can be introduced into bacteria to make medicines * Cloned genes can be used in trials of gene therapy
Steps in a cloning 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
Order the following steps in cloning a gene, placing the first step at the top. Instructions Ligation by DNA ligase produces recombinant vectors. The digested chromosomal DNA and plasmid DNA are incubated together. Chromosomal DNA is isolated and cut with a restriction enzyme; the plasmid DNA is cut with the same enzyme.
1. Chromosomal DNA ~~~ 2. The digested ~~~~ 3. Ligation by DNA ~~~~
Place in correct order the steps in constructing a cDNA library
1. Create complementary DNA using reverse transcriptase 2. Attach linkers to cDNA using DNA ligase 3. Cut the cDNAs and the vectors with the same restriction enzyme 4. Ligate the cDNAs and the vectors together 5. Transform recombinant vectors into host cells
Order the steps in DNA sequencing
1. Many copies of a single-stranded vector containing the gene of interests are acquired 2. Primers are added 3. High concentrations of all four unlabeled dideoxyribonucleotides and DNA polymerase are added 4. Low concentrations of all four fluorescently labeled dideoxyribonucleotides and DNA polymerase are added
Order the steps in performing a Western blot
1. Separate the proteins in a mixture using SDS-Page 2. Blot the proteins onto a nylon membrane 3. Incubate the nylon membrane with the primary antibody 4. Incubate the nylon membrane with the secondary antibody 5. Treat the nylon mesh with a reagent that causes a color change
What is an advantage of using cDNA in cloning?
A cDNA molecule contains the coding regions but not introns.
Why is cloning a cDNA molecule easier than cloning an entire eukaryotic gene?
A cDNA molecule does not have introns, which can be quite large.
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
What is a plasmid?
A small circular DNA molecule often used as a vector in gene cloning
Which of the following are common uses of gene cloning?
Cloned genes can be introduced into bacteria to make medicines. Cloned genes can be used in trials of gene therapy. Cloned genes can be expressed to discover their cellular functions.
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 researches to determine the order of _____ _____ in a gene
DNA nucleotides
In PCR, the template DNA is ______.
DNA to be amplified
order the steps in one cycle of a PCR reaction
Denaturation Primer annealing Primer extension
Many species of bacterial cells make restriction enzymes to protect themselves from invasion by
Foreign DNA
Which of the following is a pharmaceutical product that is produced by bacteria expressing the human gene?
Insulin
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
Polymerase chain reaction was developed by ______.
Kary Mullis
What do you call the DNA sequence in a vector that allows the replication enzymes of the host cell to make multiple copies of the vector?
Origin of replication
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.
cDNA is made using what as the starting material?
RNA
( ) DNA technology uses in vitro molecular techniques that combine DNA fragments to produce novel arrangements.
Recombinant
You have a piece of RNA, and you want to synthesize a complementary strand of DNA. Which enzyme would you use?
Reverse transcriptase
Site-directed mutagenesis allows researchers to produce a mutation at a ______ within a cloned DNA segment.
Specific site
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 many cycles of PCR, you need a machine that can change temperatures at exact times.
Thermocycler
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
How is transformation recognized?
Transformed cells can grow in conditions that untransformed cells cannot.
Which procedure is used to identify a specific protein within a mixture of many different protein molecules?
Western blotting
you wish to determine if a protein is made at a particular stage of development. What technique would you use?
Western blotting
"Sticky ends" created by cutting DNA with a restriction enzyme are useful in cloning because they ______.
are areas where two pieces of DNA can hydrogen bond
A recombinant DNA molecule has covalently linked DNA fragments from
at least two different sources
X-Gal is a colorless compound that is converted by beta-galactosidase to a dye with color
blue
If you wanted a collection of only transcribed DNA, you would construct a
cDNA library
A particular gene to be cloned is often isolated from ______.
chromosomal DNA
You would _____ a gene to make copies of that gene
clone
You would a gene to make many copies of that gene.
clone
If a gene is amplified by PCR so that there are many copies, it can be said to be ______.
cloned
Making many copies of a particular DNA segment using vectors or the polymerase chain reaction is called gene
cloning
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 consider
competent cells
Transformation occurs when
competent cells take up DNA from the medium
Transformation occurs when ______.
competent cells take up DNA from the medium
DNA made using RNA as the starting material is called
complementary DNA
In PCR, primer extension refers to the synthesis of _____ starting at the primers
complementary DNA
When DNA is made using RNA as the starting material, the DNA is called
complementary DNA
A recombinant vector ______.
contains a piece of chromosomal DNA
Single-stranded stretches of DNA created by restriction enzymes are called
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
Which of the following vectors would you use to clone a large piece of DNA?
cosmid
Vectors that can be used to clone larger segments of DNA
cosmids, BACs, YACs
Restriction endonucleases are used in gene cloning to
cut the DNA backbone prior to inserting the DNA to be cloned
Restriction endonucleases are used in gene cloning to ______.
cut the DNA backbone prior to inserting the DNA to be cloned
Molecular biologists use restriction enzymes to amplify a specific section of DNA within the genome.
false
True or false: 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
The replication of recombinant DNA molecules inside a host cell is one form of
gene cloning
A cell that harbors a vector is called a
host cell
A cell that harbors a vector is called a(n)
host cell
What is the term that describes a cell that contains a DNA cloning vector?
host cell
What does the enzyme DNA ligase do?
it covalently links the sugar-phosphate backbone of DNA
Primers are chosen for PCR based on ______.
knowing the DNA sequence flanking the gene of interest
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
When making cDNA, a short strand of DNA called a(n) might be used as a poly-dT primer.
oligonucleotide
A DNA sequence in one strand that is identical when read in the opposite direction in the other strand is called a(n)
palindrome sequence
Restriction enzymes usually recognize sequences in which the sequence in one strand is identical to the complementary strand read in the opposite direction.
palindromic
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)
plasmid
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
Short oligonucleotides that flank the region to be amplified by PCR are called
primers
If the two ends of a vector cut with a restriction enzyme ligate back together without an insert, a(n) vector has been created.
recircularized
A vector that contains a piece of chromosomal DNA is referred as a
recombinant vector
A vector that contains a piece of chromosomal DNA is referred to as a(n)
recombinant vector
Enzymes that bind to a specific DNA sequence and cut the DNA backbone are called
restriction enzymes
The enzyme ____ _____ is used when PCR is employed to detect and quantify the amount of a specific RNA
reverse Transcriptase
The enzyme that uses RNA as a template to make a complementary strand of DNA is called
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
Primer annealing occurs when
short oligonucleotides bind to complementary DNA flanking the gene of interest
Allows a researcher to produce a mutation at a specific sequence
site-directed mutagenesis
The use of dideoxyribonucleotides with different colored dyes allows the detection of
the DNA sequence
What is the purpose of Northern blotting?
to identify a specific RNA molecule within a mixture of RNA molecules
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: Chromosomal DNA is a common source of cloned DNA.
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
Many species of bacteria make restriction enzymes to protect themselves from invasion by
viruses
When using X-Gal and IPTG to differentiate recombinant from non recombinant vectors, bacteria carrying recombinant vectors are in color
white
