Genetics Ch20
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.
You are carrying out a cloning experiment and you wish to utilize the X-Gal system to identify recombinant plasmids. Into which site should you clone your DNA of interest?
lacZ gene
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
Restriction enzymes usually recognize ___________ sequences in which the sequence in one strand is identical to the complementary strand read in the opposite direction.
palindromic
In 1985, Kary Mullis developed a way to copy DNA without vectors or host cells. This technique is called ________ ________
polymerase chain reaction
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
Short oligonucleotides that flank the region of DNA to be amplified by PCR are called _____________
primers
The process by which competent cells take up DNA from the extracellular medium is called _____________
transformation
Select all vectors that can be used to clone large segments of DNA. -Plasmids -Cosmids -YACs -BACs
-Cosmids -YACs -BACs
In gene cloning, how is a suitable vector chosen?
It must replicate in the appropriate cell type
Select the reagents needed to make cDNA. -mRNA -dNTPs -Taq polymerase -Reverse transcriptase -Poly-dT primer
-mRNA -dNTPs -Poly-dT primer -Reverse transcriptase
A cell that harbors a DNA cloning vector is called a __________ _________
host cell
Viruses can be used as ____________ to carry other pieces of DNA.
vectors
In gene cloning, what is the vector?
A small DNA molecule that can replicate independently within a host cell
After digesting all of the chromosomal DNA of an organism with restriction enzymes and recombining the DNA into vectors, the collection of recombinant vectors resulting is called a __________ _________
DNA library
Naturally occurring plasmids that confer resistance to antibiotics are called __________ _________
R factors
What is the name of the enzyme that partially digests the RNA in a DNA - RNA hybrid?
RNaseH
Why would you use this RNaseH when making cDNA?
The short RNAs that result from digestion can be used as primers by DNA polymerase.
You are performing a cloning experiment with a plasmid containing an ampicillin resistance gene. You wish to use the X-Gal system to identify recombinant plasmids. What do you need to add to your media before plating the cells? -β-galactosidase -X-Gal -Ampicillin -IPTG
-X-Gal -Ampicillin -IPTG
Select the sequence that is palindromic. 5'-GGATCC-3' 3'-CCTAGG-5' 5'-AAGGAA-3' 3'-TTCCTT-5' 5'-CCACCA-3' 3'-GGTGGT-5'
5'-GGATCC-3' 3'-CCTAGG-5'
What is an advantage of using cDNA in cloning?
A cDNA molecule contains the coding regions but not introns, which can be quite large
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/genomic library
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.
You have a piece of RNA, and you want to synthesize a complementary strand of DNA. What enzyme would you use?
Reverse transcriptase
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.
What is the purpose of gene cloning?
To produce many copies of a DNA molecule of interest
"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
Many species of bacterial cells make restriction enzymes to protect themselves from invasion by __________
bacteriophages/viruses/DNA
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 many copies of that gene.
clone/amplify
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 considered _____________ cells.
competent
When DNA is made using RNA as the starting material, the DNA is called _____________ DNA.
complementary
Which of the following vectors would you use to clone a large piece of DNA? -R plasmid -Host cell -virus -cosmid
cosmid
Restriction endonucleases are used in gene cloning to ______.
cut the DNA backbone prior to inserting the DNA to be cloned
T/F: Viruses cannot be used as vectors in gene cloning. True false question.
false
The replication of recombinant DNA molecules inside a host cell is one form of ______.
gene cloning
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
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
The sequence _____________ of ______________ the of determines whether or not a vector can replicate in a particular type of host cell.
origin; replication
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 ___________ vector.
recombinant
_________________DNA technology uses in vitro molecular techniques that combine DNA fragments to produce novel arrangements.
recombinant
A molecule that has covalently linked DNA fragments from at least two sources is called a ____________ ______________molecule.
recombinant DNA
Enzymes that bind to a specific DNA sequence and cut the DNA backbone are called _______ _______
restriction; enzymes
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
Why would one use a vector with a selectable marker?
to identify cells containing the vector
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
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) _______ _______
selective marker
Which of the following is pharmaceutical product that is produced by bacteria expressing the human gene? -Insulin -Oxycodone -Aspirin -Simvastatin
insulin
When using X-Gal and IPTG to differentiate recombinant from non recombinant vectors, bacteria carrying recombinant vectors are in ______________ color
white
T/F: Chromosomal DNA is a common source of cloned DNA.
true
Which of the following are common uses of gene cloning? -Cloned genes can be used in trials of gene therapy. -Cloned genes are always used in forensic investigations. -The expression of a cloned gene can be used to discover its cellular function. -Cloned genes can be introduced into bacteria to make medicines.
-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.
Order the following 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
Place in correct order the steps in constructing a cDNA library
1. Create complementary DNA using reverse transcriptase 2. Attach linkers to cDNAs 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
