chapter 21+22 genetic
which scientist developed the polymerase chain reaction
Kary Mullis
which scientist(s) developed an early method of DNA sequencing that involved base-specific chemical cleavage of DNA
Maxam and Gilbert
'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
site-directed mutagenesis allow a researcher to make a mutation ----
at a specific sequence of DNA
a recircularized vector is one that has----
ligated itself
short oligonucleotides that flank the region of DNA to be amplified by PCR are called
primer
what activity of Taq polymerase separates the reporter from the quencher in the TaqMan molecule
5'-3' exonuclease
this figure shows a real-time PCR experiment carried out at low, medium, and high concentrations of starting template DNA. please match each curve (designated by a letter) with the corresponding amount of starting template. Fluorescence values are plotted on the Y axis.
A-high B-medium C-low
DNA made using RNA as the starting material is called ---DNA
Complementary DNA (cDNA)
naturally occurring plasmids that confer resistance are called
R factors
a recombinant vector----
a vector that contains a piece of chromosomal DNA
which of the following vectors would you use to clone a large piece of DNA
cosmid
restriction endonuclease are used in gene cloning to
cut the DNA backbone prior to inserting the DNA to be cloned
in automated sequencing, each deoxyribonucleotide is labeled with a different colored ----
fluorescent dye
in automated sequencing, each dideoxyriboneucleotide is labeled with a different colored ---
fluorescent dye
the gel retardation assay is also know as the
gel-mobility shift assay
the replication of recombinant DNA molecules inside a host cell is one form of ----
gene cloning
the enzyme--- ---- is used when PCR is employed to detect and quantify the amount of a specific RNA
reverse transcriptase
you have a piece of RNA, and you want to synthesize a complementary strand of DNA, what enzyme would you use.
reverse transcriptase
in the western blot, to what is the enzyme that will give the colored reaction coupled?
the secondary antibody
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 cell
- X-Gal - ampicillin - IPTG - b-glactosidase is expressed from the lacZ gene, not added to the media
select the type of standards that are commonly used in real-time PCR
- a standard of known concentration is added to the PCR mixture - another gene that is already present in the same sample is also amplified
order the following steps in cloning a gene, putting the first step at the top .
- chromosomal DNA is isolated and cut with a restriction enzyme, the plasmid DNA is cut with the same enzyme. -the digested chromosomal DNA and plasmid DNA are incubated together - ligation by DNA ligase
place in correct order the steps in constructing a cDNA library, beginning with the first step at the top
- create complementary DNA using reverse transcriptase - attach linkers to cDNA using DNA ligase - cut the cDNA and the vectors with the same restriction enzyme - ligate the cDNAs and the vectors together - transform recombinant vectors into host cells
order the steps in one cycle of a PCR reaction, putting the first step at the top
- denaturation - primer annealing - primer extension
Steps of Northern Blotting
- extract and purify RNA from living cells - load RNA onto an agarose gel - separate RNA molecule according to size - blot RNAs onto a nylon membrane probe with a labeled fragment of DNA - probe with a labeled fragment of DNA
select the reagents needed to make cDNA
- reverse transcriptase - dNTPs - mRNA - Poly-dT primer
which of the following are 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 trails of gene therapy
when the CRISPR-Cas system is used for gene mutagenesis, which two components are combined in the single guide(sgRNA)?
- tracrRNA -crRNA
order the steps in DNA sequencing, putting the first step at the top
1, many copies of a single-stranded vector containing the gene of interesting are acquired. 2. primers are added 3. high concentrations of all four unlabeled deoxyribonucleotides are added 4. low concentrations of all four fluorescently labeled dideoxyribonucleotides and DNA polymerase are added 5, electrophoresis separates DNA according to their length 6. a laser and fluorescence detector are used to determine the color associated with each DNA strand
place the steps in a real-time PCR experiment in order from first to last, putting the fist step at the top
1. a primer and the taqman probe both anneal to template DNA 2. taq polymerase cleaves the oligonucleotide 3. the reporter can emit unquenched fluorescence that can be measured 4. more and more taqman probes digested and the level of flurescence increases
oder 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 cur with the same enzyme 2. the digested chromosomal DNA and plasmid DNA are incubated together. 3. ligation by DNA ligase
place the steps in site-directed mutagenesis in order from first to last, with the first step on top
1. design an oligonucleotide primer that is complementary to the DNA of interest, except for a mismatched region 2, anneal the primer to the template DNA 3, add dNTPs, DNA polymerase, and DNA ligase. 4. introduce the DNA into a live cell
order the steps in performing a western blot, putting the first step at the top
1. separate the proteins in a mixture using SDS-PAGE. 2. blot the proteins onto a nylon membrane.n 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
a researcher may use restriction enzymes to digest the DNA of an organism. then fragments of DNA are then legated individually into many vectors. this collection of recombinant vectors is called a---- ----
DNA library
when cloning gene into a vector, the sugar-phosphate backbone of each DNA molecule is covalently linked by the enzyme DNA----
DNA ligase
This technique enables researchers to determine the DNA bases in genes and other chromosomal regions
DNA sequencing
which technique can identify the DNA region that interacts with a DNA-binding protein?
DNase I footprinting
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
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 ---
bacterial phases
X-Gal is a colorless compound that is converted by beta-galactosidase to a dye with ---- color
blue
how does one analyze a site-directed mutation
by introducing it into a living organism
A DNA library made with DNA generated by reverse transcriptase is called a ----library
cDNA
in a 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
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
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 cells
transformation occurs when---
competent cells take up DNA from the medium - the ligation of a gene into a vector is not the same as transformation, the uptake of extracellular DNA into a cell
reverse transcriptase PCR can be used to ---
detect and quantify the amount of a specific RNA - RNA molecules are amplified into DNA in this process, regardless of the presence restriction sites
the DNA sequencing method developed by Fredrick Sanger that became a commonly used method of DNA sequencing is called --- sequencing
dideoxy sequencing
if the oxygens on carbon2 and 3 of the sugar of the nucleotide have been removed, the nucleotide is referred to as a
dideoxyribonucleotide
the early mammalian embryo contains ---- ---- cells, which are pluripotent cells found in the inner cell mass of the blastocyst
embryonic stem
which fluorescences under UV light and can be used to detect DNA in a gel
ethidium bromide
true or false: plasmids are the only appropriate vectors for generating a genomic library.
false
true or false: viruses cannot be used as vectors in gene cloning
false - viruses can be used as vectors, typically to carry small pieces of DNA
what is the term that describes a cell that contains a DNA cloning vector?
host cell
in a palindromic sequence, the sequence in one strand ----
is the same in the complementary strand when read in the opposite direction
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 - the 3' end of mRNA has a poly-A tail
a recircularized vector is one that has
ligated with itself
a major advantage of CRISPR-Cas technology over site-directed mutagenesis is that it can be used directly on--- cells
living
place the characteristic of gene mutation by the CRISPR-Cas system in the correct category based on repair mechanism. - nonhomologous end-joining - homologous recombination repair
non-homologous end-joining -the break region may incur a small deletion - frameshift may inactivate the gene homologous recombination repair - researcher includes donor DNA that is homologous to region where break occurs - used to make s specific point mutation
how can PCR amplify one segment of DNA from a complex mixture of potential template molecules?
northern blotting
the technique is used to identify a specific RNA molecule within a mixture of RNA molecule
northern blotting
site-directed mutagenesis is sometimes referred to as ---- directed mutagenesis
oligonucleotide-directed mutagenesis
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
origen of replication
a vector must contains 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
A DNA sequence in one strand that is identical when read in the opposite direction in the other strand is called a ----sequence
palindromic
a --- is a small circular DNA molecule that can replicate with bacterial cels and is often used as a vector in gene cloning.
plasmid
during RCR, the process of --- --- results when the Taq polymerase catalyzes the synthesis of complementary DNA, starting at the primer
primer extension
the DNase I footprinting technique is used to study---- ----interaction
protein-DNA interaction
---- ---- PCR allows one to assess the amount of DNA produced during a PCR amplification as it is happen
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
----DNA technology uses in vitro molecular techniques that combine DNA fragments to produce novel arrangements.
recombinant
a resistance gene that allows a host cell containing a vector to grow on a toxic substance is called a(n) ---- marker
selectable marker
the dideoxy sequencing method results in mixtures of DNA strands of different lengths which are run on a gel is read from the bottom up. this is referred to as a
sequencing ladder
primer annealing occurs when
short oligonucleotides bind to complementary DNA flanking the gene of interest
when the CRISPR-Cas system is used for gene mutagenesis, tracrRNA and crRNA are linked together in a molecule called the ---- ---- RNA
single guide RNA
if a scientist wanted to determine how a specific mutation in a gene of interest affected an organism, what technique would be most useful?
site-directed mutagenesis
single-strand stretches of DNA created by restriction enzymes, such as those shown on the lefty and right sides of the molecule in the figure, are called ---- ----
sticky ends
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
in PCR, the template DNA is---
the DNA to be amplified - the gene of interest
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.
which sequence determines whether or not a vector can replicate in a particular cell
the origin of replication
in the western blotting, what binds to the protein of interest?
the primary antibody - the secondary antibody bonds to the primary antibody
RHaseH partially digests the RNA in a DNA-RNA hybrid molecule. why would you use this enzyme when making cDNA?
the short RNAs that result from digestion can be used as primers by DNA polymerase.
to perform PCR, a machine called a --- automates the timing of each cycle
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 f the gene of interest?
they are complementary to the flanking sequence
how does a bacterial cell use restriction enzymes?
to protect the cell against invasion by bacteriophages
the natural function of the CRISPR-Cas system in bacteria to ----
to provide defense against bacterio phages
the process by which competent calls take up DNA from the extracellular medium is called---
transformation
the process by which competent cells take up DNA from the extracellular medium is called
transformation
what is the term for an organism that has received genetic material from a different species?
transgenic organsim
true or false: chromosomal DNA is a common source of cloned DNA
true
true or false: reverse transcriptase PCR can detect very small amounts of a specific RNA.
true
a small DNA molecule that can replicate independently within a host cell and thus make many copies of an inserted gene is called
vector
viruses can be used as --- to carry other pieces of DNA
vectors
you wish to determine if a protein is made at a particular stage of development. what technique would you use
western blotting - this method can determine if a specific protein is made in a particular cell type or at a particular stage of development
