CH 20
Cells that can take up DNA from the medium are consider
competent cells
cDNA is made using what as the starting material?
RNA
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
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
( ) DNA technology uses in vitro molecular techniques that combine DNA fragments to produce novel arrangements.
Recombinant
What does the enzyme DNA ligase do?
it covalently links the sugar-phosphate backbone of DNA
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
Site-directed mutagenesis allows researchers to produce a mutation at a ______ within a cloned DNA segment.
Specific site
You would _____ a gene to make copies of that gene
clone
The replication of recombinant DNA molecules inside a host cell is one form or
gene cloning
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
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
A molecule that has covalently linked DNA fragments from at least two sources is called a
Recombinant DNA molecule
To perform many cycles of PCR, you need a machine that can change temperatures at exact times.
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 Southern blotting?
To identify a particular DNA sequence within a mixture of DNA fragments
How does a bacterial cell use restriction enzymes?
To protect the cell against invasion by bacteriophages
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
If you wanted a collection of only transcribed DNA, you would construct a
cDNA library
Making many copies of a particular DNA segment using vectors or the polymerase chain reaction is called gene
cloning
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
When DNA is made using RNA as the starting material, the DNA is called
complementary DNA
A vector requires an origin of replication so that it can be
copied many times by the host cell
Vectors that can be used to clone larger segments of DNA
cosmids, BACs, YACs
Molecular biologists use restriction enzymes to amplify a specific section of DNA within the genome.
false
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
Short oligonucleotides that flank the region to be amplified by PCR are called
primers
A vector that contains a piece of chromosomal DNA is referred as a
recombinant vector
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
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
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
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
Restriction endonucleases are used in gene cloning to
cut the DNA backbone prior to inserting the DNA to be cloned
What is a plasmid?
A small circular DNA molecule often used as a vector in gene cloning