Genetics Ch 20
The 2 important genes vector carries
1. ampR 2. lacZ
Which component in the CRISPR-Cas9 system makes a double-strand break in DNA?
Cas9
Genes are cloned by splicing a fragment of _____ from an organism into a cloning vector to make a recombinant DNA molecule
DNA
A collection of recombinant vectors
DNA library
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 vector is called a ______ ______
DNA library
After ligation, there is a collection of recombinant vectors, with each vector containing a particular fragment known as a ____ ______
DNA library
DNA sequencing enables reseacrhers to determine the order of ______ _____ in a gene
DNA nucleotides
Dideoxy sequencing was formulated based on scientist knowledge of what process?
DNA replication
This technique enables researchers to determine the DNA bases in genes and other chromosomal regions
DNA sequencing
The DNase I footprinting technique is used to study _______-_____ interactions
DNA-protein
The most common plasmids are the ___ ____ plasmid cloning vectors
E. coli
Director of Human Genome Project
Francis Collins
Which scientist developed the polymerase chain reaction?
Kary Mullis
If we want only one region of genomic DNA or one mRNA then we use _______ ________ _______
Polymerase Chain Reaction
_______ _____ occurs when short oligonucleotides bind to complementary DNA flanking the gene of interest
Primer annealing
____ _____ _____ is used to quantitate the amount of a specific Target DNA
Real-Time PCR
The use of in vitro molecular techniques to isolate and manipulate fragments of DNA
Recombinant DNA technology
Cuts the phosphate-sugar backbone between the phosphate and the 3' carbon
Restriction Enzyme
Multiple cloning site = ______ ______ _____
Restriction site cluster
____-______ ________ alows a reseacher to produce a mutation at a specific sequence
Site-directed mutagenesis
In PCR, each cycle uses the products of the previous cycle as a template. What do you call this?
a chain reaction
In PCR, each cycle uses the products of the previous cycle as templates. What do you call this?
a chain reaction
Confers antibiotic resistance to the host cell and identifies cells that have taken up the vector
ampR
If the starting material was cDNA, the library is called a _____ _______
cDNA library
An actual ______ library would contain thousands of different bacterial colonies. Each one carrying a different piece of cDNA created from the starting _____ sample
cDNA; RNA
Oligonucleotides called linkers, with a restriction enzyme site, are attached to the ______ using ____ _______
cDNAs; DNA ligase
A particular gene to be cloned is often isolated from ______ _____
chromosomal DNA
To make many copies of a gene, you would _____ that gene
clone
A DNA molecule that acts as a carrier of DNA that is to be cloned
cloning vector
A molecule that carries the DNA to be cloned
cloning vector
Cells that are able to take up DNA
competent cells
In PCR, primer extension refers to the synthesis of _________ _____ starting at the primers
complementary DNA
The DNA sequencing method developed by Frederick Sanger that became a commonly used method of DNA sequencing
dideoxy sequencing
The gel retardation assay is also know as the ______ ____ _____ _____
electrophoretic mobility shift assay
What technique is useful for studying protein-DNA interactions?
electrophoretic mobility shift assay and DNase I footprinting
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
The amount of product nearly doubles with each cycle but may be difficult to detect as the amounts are small
exponential
In automated sequencing, each dideoxyribonucleotide is labeled with a different-colored _______ _____
fluorescent dye
The TaqMan system uses a detector oligonucleotide that has a _______ _______ _______ at one end and a ________ ______ at the other end
fluorescent reporter molecule; quencher molecule
The process that produce many copies of a DNA moledcule of interest
gene cloning
If the starting material was chromosomal DNA, the library is called a ______ _______
genomic library
An actual ______ library would contain thousands of different bacterial colonies. Each one carrying a different piece of ________ _____
genomic; chromosomal DNA
Very active Ori, generates a _____ copy number per cell
high
A cell that harbors a vector
host cell
Recombinant DNA molecule is introduced into a _____ _____ where it is replicated aka ________
host cell; transformation
Encodes β-galactosidase and provides a means by which bacteria that have picked up the cloned gene can be identified
lacZ
In the hybrid vector, the chromosomal DNA inserts into the ____ gene, thereby disrupting it
lacZ
Accumulation of the product shows a directly proportional relationship to cycle number
linear
In PCR, the temperature must be _______ from the denaturation temperature in order for primers to anneal
lowered
This process indentifies a specific RNA moledcule within a mixture of RNA molecules
northern blotting
Human chromosomal DNA generate thousands of fragments. Typically ____ fragment inserts into a vector and becomes a ________ _______
one; recombinant vector
Allows for replication of plasmid in E. coli
ori sequence
A vector requires an _____ ___ ______ so that it can be copied many times by the host cell
origin of replication
What do you call the DNA sequence in a vector that allows that replication enzymes of the cell to make lots of copies of the vector?
origin of replication
________ means the base sequence reads the same backwards and forwards.
palindromic
A small circular DNA moledcule that is often used as a vector in gene cloning
plasmid
Transformation refers to uptake of _______ vectors by a ______ cell
plasmid; bacterial
Plasmids used for cloning experiments are derived from naturally occurring ______ that have been modified to facilitate cloning
plasmids
Accumulation of the product level off as one or more reagents are used up
plateau
In 1985, Kary Mullis developed a way to copy DNA without vector or host cells. This technique is called _______ ______ _____
polymerase chain reaction
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 amplifies by PCR
primers
When using PCR to amplify DNA, short oligonucleotides called ________ flank the region of DNA to be amplified
primers
What is the technique that allows one to determine the amunt of template DNA present when the PCR cycles began?
quantitative PCR
_______-_____ PCR allows one to assess the amount of DNA produced during a PCR amplification as it is happening
real-time
In contrast to the hybrid vector, the _________ vector has a functional ____ gene
recircularized; lacZ
The production of new arrangements of DNA
recombinant DNA technology
The use of in vitro molecular techniques that combine DNA fragments to produce novel arrangements
recombinant DNA technology
A vector that contain a piece of chromosomal DNA
recombinant vector
Vector that contains a piece of chromosomal DNA
recombinant vector
ampR
resistance to ampicillin
camR
resistance to chloramphenicol
tetR
resistance to tetracyclin
Enzymes that bind to a specific DNA sequence and cut the DNA backbone
restriction endonucleases
_______ ______ are used in gene cloning to cut the DNA backbone prior to inserting the DNA to be cloned
restriction endonucleases
Site-directed mutagenesis allow a researcher to make a mutation at a ________ _____ of DNA
specific sequence
In PCR, the DNA to be amplified is called the _______ DNA
template
In PCR, the _____ DNA is the DNA to be amplified
template
Treating chromosomal DNA with restriction enzymes can generate _____ ___ _______ of fragments
tens of thousands
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
Use a ______ _________ (essential equipment) to generate different temperatures for each cycle
thermal cylcer
True or False: Chromosomal DNA is a common source of cloned DNA
true
A small DNA molecule that can replicate independently within a host cell
vector
A small DNA molecule that can replicate independently within a host cell and thus make many copies of an inserted gene
vector
Transfection refers to introduction of ______ vectors into a ____ _____
viral; host cell
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
An E. Coli Cloning Vector Must Have Three Features
1. Ori sequence 2. Selectable marker 3. restriction enzyme cleavage sites
The enzyme _______ ________ is used when PCR is emplyed 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
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
_______ ______ PCR can be used to detect and quantify the amount of specific RNA
reverse transcriptase
The use of dideoxyriblonucleotides with different colored fluorescent dyes allows the detection of the _______ ___ ____
sequence of DNA
To perform PCR, a machine called a _________ automated the timing of each cycle
thermocycler