Recombinant DNA Flashcards
____ is producing exact copies of a DNA segment
cloning
_____ _____ is the ____ _____ in which foreign DNA is inserted for cloning
cloning vector, DNA molecule
What are the 4 common types of cloning vectors?
plasmids, cosmids, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YACS)
Cloning vectors with their inserted foreign DNA can be introduced into host cells where they are able to _____
multiply
Is cloning in vitro on in vivo?
in vivo
Is PCR in vitro or in vivo?
in vitro
In vivo means occurring where?
in a host cell
In vitro means occurring where?
in a test tube
What cloning vectors can fit the largest size of DNA insert?
YAC
What cloning vector fits the smallest size of DNA insert?
plasmid
Foreign DNA fragments are inserted into the ____ ____ ______
plasmid cloning vectors
The plasmid vector and foreign DNA are treated with a _____ _______
restriction endonuclease
_____ ______ is the enzyme that cuts DNA at a specific sequence
restriction endonuclease
Restriction endonuclease creates ____-______ ends when cutting
single-stranded
Single-stranded ends are useful for what two purposes in cloning?
base-pairing of the foreign DNA and plasmid vector, resealing the backbone by linking the backbone of the foreign DNA and plasmid (a covalent linkage)
_____ is the term for the base-pairing of the single-stranded ends
annealing
Ligating is the _____ linking of the backbone of the foreign DNA and the plasmid vector
covalent
The recombinant DNA has a _____ circumference than the original plasmid
larger
The base-pairs of the foreign DNA ____ bond with the base pairs of the plasmid vector
Hydrogen
A _____ plasmid is one with partly foreign DNA And partly plasmid SNA
chimeric
The DNA backbones of the plasmid vector and the foreign DNA ____ link with each other
covalently
A ____ _____ is a set of cloned chromosomal DNA fragments representing the entire genome of an organism
genomic library
Isolating DNA, treating DNA with a specific enzyme to be cut into fragments, producing fragments 4,000 bp in length, and placing the fragments into unique plasmids is the process used to produce ____ _____
genome libraries
A ____ _____ is a set of cloned cDNAs representing all of the mRNA (gene transcripts) synthesized by cells or tissues
cDNA library
cDNA is useful for ____ ____ because it does not have ____
protein expression, introns
______ are DNA copies of mRNA molecules
cDNAS
The primer connects to the ________when synthesizing cDNA molecules
poly-A tail
____ ______ uses RNA sequences to copy into DNA and adds new DNA nucleotides during cDNA synthesis
reverse transcriptase
The heteroduplex is the hybrid ___/____ molecule
DNA RNA
_______ attacks RNA to remove it when making cDNA
RNase H
___ ______ copies first strand cDNA using RNA segments as primer
DNA polymerase
When making cDNA, DNA fragments are joined by ___ _____ to make the end helix
DNA ligase
A vector for in vitro expression of a cDNA molecule or other foreign DNA can be constructed by placing a ____ _____ next to the ___ _____
promoter sequence, inserted DNA
In RNA expression, the host ___ _______ bonds to the plasmid with foreign DNA to produce mRNA
RNA polymerase
To express a protein in a host cell, a cDNA molecules is inserted next to a ___ _____ and a _____-____ ______
promoter sequence, ribosome-binding site
A _____ ______ ______ is a sequence of 6 restriction enzymes that are the recognition and cutting sites and is where you cleave the plasmid to insert DNA
polylinker cloning site
The mRNA produced via protein expression vector contains the ____-____ _____ but not the promoter
ribosome-binding site
The benefit of producing protein via protein expression vector is that cells can later be harvested and the proteins purified and what?
cells can be frozen and stored to be used later to restart protein synthesis
PCR is used to _____ a specific _____ ______
amplify, DNA segment
The 3 steps to PCR are ____, ____, and _____
separating, annealing, extending
PCR ______ occurs at the highest temp
seperating
PCR primers are ____
oligonucleotides
The target DNA for PCR does/does not need to be purified
does not
The amount of DNA _____ with each cycle of PCR
doubles
DNA polymerase are heat _____
sensitive
What is the DNA polymerase that is heat sensitive and used in PCR?
taq polymerase
___ ______ eliminates the need for fresh DNA polymerase for each cycle of PCR
Taq polymerase
___ ______ cut DNA at specific sequences and have subsequently been used for cloning and sequencing of DNA molecules
restriction endonucleases
If the res. enzyme cuts an even number sequence, they are _____
palendromes
Res. enzymes that make staggered cuts produce _____-_____ ends
single strand
Res enzymes that make _____ cuts produce single-stranded ends
staggered
Res enzymes that make non-staggered cuts produce ____ ends
blunt
Rest enzymes that make ____-____ cuts produce blunt ends
non-staggered
_____ end cuts are less useful in cloning
blunt
_____ cuts go down the middle
blunt
___ ______ is the blotting method where DNA fragments are separated by gel electro., transferred to a sheet of membrane, and detected with a probe
southern blotting
___ ____ is the blotting method where RNA molecules (such as mRNAs) are separated in a gel, transferred to a sheet of membrane, and detected with a probe
northern blotting
____ _____ is the blotting method where proteins are separated in a gel, transferred to a sheet of membrane, and detected with antibodies
western blotting
Southern blotting is used for ____ detection
DNA
Northern blotting is used for _____ detection
RNA
Western blotting is used for ____ detection
protein
If the desired protein is detected in western blotting, it will bind to the _____
antibodies
____ ____ ______ is the introduction of a functional gene into cells of human tissues and organs to correct damage caused by a mutant gene
human gene therapy
Human gene therapy is used to produce _____ _____ in ___ ______
human protein, human cells
An ___ ______ consists of a cDNA version of a gene and a promoter for human gene therapy
expression cassette
In gene therapy, the expression cassette is incorporated into a _____ that can infect human cells
virus
Viral vectors for human gene therapy include ____ ____ ____ and _____ ______
DNA virus vectors, retrovirus vectors
Human gene therapy occurs in vivo or in vitro?
in vivo
The DNA virus used in human gene therapy is the _______
adenovirus
Adenovirus vectors are produced by deleted __ ____ to create space for the ___ _____
gene E1, expression cassette
The complementing cell line contains the E1 gene which allows the adenovirus vector to ____ and become _____ into infectious virus
replicate, incorporated
The adenovirus from the complementing cell line is used to ____ the cells of a patient and express the ___ _____
infect, functional protein
Cystic fibrosis, sickle-cell, hemophilia, and muscular dystrophy are examples of what type of disease?
single-gene
Rank the sizes of the DNA cloning vectors: YAC, plasmid, BAC, cosmid
plasmid<cosmid<BAC<YAC
cDNAs are DNA copies of _____ molecules
mRNA
The 3 steps to PCR are ____, ____, and ____
separate, anneal, extend
In PCR, what component of the reaction mixture is altered to introduce a mutation into the nucleotide sequence of a gene that is to be amplified?
primers
