Molecular Genetics Flashcards
the use of molecular genetics revolution techniques to develop mew products is called
Biotechnology
a set of molecular techniques for locating, altering, recombining and studying specific pieces of DNA is called
Recombinant DNA
an example of restructuin enzymes
site-specific endonucleases
what method are designed to cleave DNA at predetermined sequences
Engineered nucleases
what is a facile method for ediiting genomes across the phylogenetic spectrum
CRISPR-Cas genome editing
locating DNA fragments can be done with
hybridization probes
what enzymes are responsible for recognizing and cutting DNA at specific nucleotide sequences
Restriction enzymes
what is the most useful class of restriction enzymes
Type II
Restriction enzymes are part of _________ restriction modification system which is a defense against viruses
bacterium
each restructuin enzyme is paired with which enzyme that methylates the recognition sequence
restriction methylase
function of methylation of recognition sequence
blocks cleavage by the restriction enzyme to protect the bacterium’s own DNA from being digested
what does the first 3 letters of the abbreviation for each restriction enzyme mean
refers to the bacterial species from whcih the enzyme was isolated (Eco for E.coli)
what does the 4th letter of the restriction enzyme refer to
the strain of bacteria from which the enzyme was isolated
what doe sthe roman numerals that follow the letters mean
identify different enzymes from the same spe cies
restriction sites thend to be how long and generally
about 4-8 bp long and generally palindromic
the ends of restruction enzymes produced by cleavage are of which types
Cohesive ends ( 5’ overhangs or 3’ overhangs)
Blunt ends
overhangs are associated with which end
cohesive ends
even length ends from both single strands are which end
blunt end
the enzyme BamHi is from which microorganism
A. Clostridium formicoaceticum
b. Haemophilus aegyprius
c. Bacillus amyloliquefaciens
c. Bacillus amyloliquefaciens
how do restriction enzymes producing blunt ends and cohesive ends in DNA
by making double stranded cuts
cohesive ends are also known as
sticky. ends or overhanging ends
the nicks in the sugar phosphate backbone of two fragments can be sealed by
ligase
is it possible to insert DNA into a complex genome at single predetermined site using convention restriction enzymes?
NO
CRISP-CAS immunity is in
bacteria and archae
CRISP RNA are encoded by
Clustered Regularly Interspersed Short Palindromic Reoeats
A CSRISPR array is derived form
bacteriophage and plasmid genomes
______ combine with Cas protein to provide defense against invasion by specific foreign DNA molecules
CRSIPR RNAs
After expression and processing what happens to the foreign DNA
crRNA will join Cas to form an effector complex that binds to foreign DNA through base pairing
what makes the DNA nonfunctional after expression and processing
when the Cas protein cleaves to the foreign DNA
CRISPR-Cas 9 is a technique for ______
precisely editing the genome
advantages of CRISP-Cas 9
- can be used in intact cells
- can be used in many species, including humans
- relatively long sequence recog. by sg RNA (Edits can happen almost everywhere in genome
- easier and less expensive than altering DNA binding protein
- modified to introduce single cuts and sticky ends
having single-stranded cuts and sticky ends through crisp-cas 9 means
improves efficiency of inserting DNA fragments
Waht are some limitation and challenges of CRISPR-Cas 9
potential for off-target cleavage
genetic mosaic may result when applied to multicellular embryos
difficulty introducing the system into cells
is Cas9 100% EFFICIENT
NO, some cells are edited while others are not
Concerns with use of CRISPR-Cas 9 editing
- could be modifying humans in questionable ways
- already been used on human embryos but they did not complete development
- germ line editing allows traits to be passed down through generations
- Safety concerns because of off target cuts
- potential danger of editing animals and plants that are released into the wild
what method is used to seperate DNA molecules on the basis of their size
Gel electrophoresis
after gel electrophoresis, DNA fragments appear as
bands on the gel
which method can detect a single restriction fragment in a complex mixture of restriction fragment
Southern blotting
in southern blotting, what DNA fragment will give signal on an autoradiogram
the fragments hybridized to a labelled probe
what are the types of gel blots
- SOuthern blots
- Northern blots
- Western blot
in which gel blot, is the DNA on the gel transferred to a filter then hybridized with a DNA or RNA probe
Southern blot
in which gel blot, is the RNA on the gel transferred to a filter then hybridized with a DNA or RNA probe
northern blot
in which gel blot is the protein on the gel transferred to a filter, an antibody is bused to detect a specific protein
Western blot
specified DNA fragments can be amplified through
PCR (Polymerase Chain reaction),
molecular cloning
what is an application of PRC
real time PCR
the process of quantitavely detrmining the amount of DNA amplified as the reaction proceed is called
Real time PCR
What are the limitation of PCR
- Sequence of the target DNA must be known2.
- Amplification of contaminants
- Accuracy
- Length of the PCR products
in molecular cloning, how is a specific piece of DNA amplified
using a host cell
what were used to replicate the desired piece of DNA in the host cell
Cloning vectors
Examples of gene cloning
plasmid vectors
transformation
screening cells for recombinant plasmids
other cloning vectors
genetic engineering of plants for inset resistance
circular DNA molecules from bacteria are called
Plasmids
how is foreign DNA inserted into plasmid
using restriction enzymes and DNA ligase
what are synthetic DNA fragments containing restriction sites
linkers
what are used to confirm whether or not the cells have been transformed
Selectable markers
an ideal cloning vector must have
- Origin of replication recognized in the host cell
- Selctable marker
- One or more unique restriction sites
pUC19 is an axmple of
cloning vector
what gene is used to screen for bacteria containing recombinant plasmids
LacZ gene
original plasmid (nonrecombinant) produces which enzyme
Beta-galactosidase which cleaves to X-gal,
what does the colonies of bacteria with a recombinant plasmid looks like
they remain white because recombinnat plasmid cannot synthesize Beta-galcatosidase
what causes colonies to turn blue
beta-galctosidase produces by original plasmid which cleaves to X-gal
examples of cloning vectors
phages
cosmids
Bacterial Artifical Chromosomes (BACs)
Yeast Artifical Chromosomes
Ti plasmids
to ensure transcription and translation, a foreign gene may be inserted into
an expression vector like E-coli
what contains operon sequences that allow inserted DNA to be translated and transcribed, and also sequences that regulate
expression vector
gene-encoding repressor in expression vector does what
bind O (operator and regulates P(bacterial promoter sequences)
What plasmid from Agrobacterium can be used to transfer genes into plants
Tiplasmid
what is a gram-positive bacterium that produces several substances toxic to insects
Bt (Bacillus thuringiensis)
Bt toxicity is specific to_______ and expressed in _____ to produce ______
insects, plants, insect resistant plants
most notable insecticidal chemicals in Bt are
crystalline protein (cry called DElta-endotoxins)
the Bt ttoxin gene was isolated from _____ and transferred to ____
bacteria, transferred to tobacco plants
Transgenic tobaccos expressing Bt is protected from damage by ____
tobacco hornworm larvae
DNA hybridization probes can be used to locate the
chromosomal or cellular location of a gene or its RNA product
what is cDNA
DNA copy of mRNA
how is cDNA made double stranded
DNA Polymerase
