Lecture 9 Biotechnology Flashcards
What is polymerase chain reaction
The presence of a DNA segment is detected and the size is determined
What does colony blotting do?
Detects specific DNA sequence in colonies grown on agar plates
What cuts DNA strands at recognition sequence
Restriction
Restriction enzymes generate ______
restriction fragments
Complementary sticky ends can ______
Anneal
What does anneal mean
Form base pairs with one another
DNA ______ joins molecules covalently
ligase
Restriction enzymes allow creation of ______ DNA molecules
Recombinant
Restriction enzymes recognize a ______ to ______ base pair nucleotide
4 - to 6-
palindromes
The same on both strands when read in 5’ to 3’
What does gel electrophoresis do
Separates DNA fragments by size
What does the current in the electrophoresis do/
causes DNA to migrate through gel to + electrode
In gel electrophoresis, smaller fragments move ______
faster
What does CRISPR system do
Locates and alters specific sites in DNA molecules
What is the DNA cutting enzyme and what is it used for
cas9, gene editing
How does cas9 cut DNA?
uses single stranded RNA to recognize DNA
Makes double stranded cut at DNA
DNA can be altered by manipulation DNA repair mechanism
dcas9 (dead) does not cut DNA but still ______
uses RNA guide to bind to specific DNA sequence
dCas9 may block ______
RNA polymerase
dCas 9 may deliver molecules to certain ______ locations
Chromosome
dCas9 can deliver a ______ marker to locate a gene on chromosome
Fluorescent
What is genetically engineered bacteria useful for
Protein production, DNA production, Research tools
CRISPR cas9 can genetically engineer bacteria, but most engineering uses ______
DNA cloning
How does DNA cloning work
isolate DNA
cut with ______ enzyme
join DNA fragment with ______ to generate recombinant molecules
introduce into host where it will ______
high copy number vector makes large amounts of ______
restriction
vector (plasmid)
replicate
protein
Why is genetically engineered bacteria better than other sources of bacteria?
Safer than taking from animal, won’t cause allergic reactions
How is genetically engineered bacteria used in research
Clone DNA segment into bacterium
allows easier production of DNA for study
Fewer genes in bacterial cell
A plant or animal with a cloned gene is ______
transgenic
What is DNA library
Collection of clones that contain the entire genome together
How do you create a DNA library
Cut DNA with restriction enzyme
clone all fragments into population of E.Coli
Before cloning eukaryotic genes into bacteria, ______ must be removed
Introns
How does a researcher obtain a bacteria without introns
Isolate mRNA
reverse transcribe to single DNA strand
Synthesize complementary strand to form double stranded cDNA
When obtaining bacteria without introns, the complementary strand is synthesized to form double stranded ______
cDNA
When generating a recombinant DNA, vector is usually a modified ______
plasmid or bacteriophage
the vector has origin of replications and carries ______
Cloned DNA
The vector must have ______ sites where DNA is cut so that insert can be joined to it
restriction
______ site recognizes insert cut
multiple cloning
Vector has ______ marker
selectable
- only cells that have vector sequences will grow
Second marker is disrupted by ______ when insert is present
Insertional inactivation
What does insertional inactivation do
distinguishes recombinant plasmids from intact vector
vector ______ selects for cells with vector and differentiates those with recombinant plasmids
pUC18
What is the selectable marker
Ampicillin resistance
What is the second genetic marker
lacZ’ gene
What does the product of the lacZ’ gene do?
cleaves X-gal, forms blue compound
What do the intact vectors become?
blue colonies
What do the recombinant molecules become?
White colonies
What does DNA sequencing do?
Determines the order of nucleotides in a DNA molecules
What type of synteshis is the Dideoxy chain termination method?
In vitro DNA synthesis
What does the dideoxy chain termination method require?
Template DNA, DNA polymerase, Primer, neoxynucleotides, dideoxynucleotides
in dideoxy chain termination, ddNTP act as ________
Chain terminators
What does ddNTP lack
3’OH
Synthesis stops when ________ is incorporated
ddNTP
in Dideoxy chain termination, different ddNTPs carry ________
Distinct fluorescent marker
in Dideoxy chain termination, laser reads ________ labels on separated ddNTPs
fluorescent
how is intensity shown by the laser in DCT?
Recorded as a peak
In DCT, the order of the color peaks reflect what?
Nucleotide sequence of DNA
What is high-throughput sequencing?
high automated methods that generate huge amounts of DNA sequence quickly
What does high-throughput sequencing use to form result?
Millions of overlapping small sequences
What aligns and merges the data for analysis in high-throughput sequencing?
Computers
What does nanopore sequence measure?
brief changes in electrical current to determine nucleotide sequence of long fragments as it passes through microscopic pores in membrane
in nanopore sequencing, long fragments of ________ are in electrically conductive solution
ssDNA
Nonopore sequencing is when long fragments pass through microscopic pores where different nucleotides ________ electrical currents to varying degrees
block
In nanopore sequencing, recorded ________ reflect nucleotide sequence
disruption
What sequencing method is used to sequence microbial DNA on international space station?
Nanopore
Polymerase Chain Reaction allows for how many DNA?
Creation of over a billion copies of DNA in hours
PCR product can be visualized through ________
Gel electrophoresis
PCR can detect ________ without culturing
Pathogens
What is RT-PCR
Reverse transcription PCR
RT-PCR is used to synthesize ________ from mRNA template within a sample
cDNA
What is cDNA template for
Amplification
What is qPCR
Quantitative PCR
qPCR labels the PCR product with ________
Fluorescent
Why does qPCR mark the PCR with fluorescent marker?
It’s accumulation can be monitored by the marker, which is shown but the amount of fluorescence
how does qPCr track the amplification?
in real time
What is qPCR primarily used for
Amplification
qPCR determines what?
The amount of target DNA in a sample
The process of DNA synthesis requires a ________ stranded DNA with a ________ DNA
double, template
The process of DNA synthesis requires ________ polymerase
Taq
What is Taq polymerase
heat-stable DNA polymerase from Termus aquaticus
Process of DNA synthesis requires ________ to determine which portion of the template DNA will be amplified
Primers
The process of DNA synthesis requires ________
Deoxynucleotides
What are the 4 deoxynucleotides used in DNA synthesis
dATP, dGTP, dCTP, dTTP
What is the first step of the amplification cycle in PCR
DNA is denatured by heating, approx. 95 degrees
What is the second step of amplification cycle of PCR
Temperature lowered to allow primers to anneal
What is the third step of amplification of PCR
Temperature raised to allow DNA synthesis
In amplification cycle, target DNA is ________ in each cycle
Doubled, exponential cycle
In amplification cycle, what is an important enzyme and why?
Taq polymerase because it is heat-stable and doesn’t denature in the step that DNA does
________ produce PCR product containing only target sequence
Primers
Primers produce PCR product with only ________
Target DNA
Primers require how many cycles to produce PCR product?
3
What does the sequence of primers determine in PCR?
The length of DNA amplified
in PCR, there is ________ amplification of DNA
Exponential
in PCR, there is ________ numbers of product of correctly defined length
increasing
What are STRs?
Short tandem repeats
What does STRs do?
Amplifies certain chromosomal regions
STRs repeat within
intron or untranslated region
What does the CODIS database contain
Amplification patters of 20 different STR loci (Chromosomal locations) and allows unique identification
What do DNA probes do?
Locate specific nucleotide sequence
Probe is a ________ DNA piece that is ________ to sequence of interest
single stranded, complementary
DNA probes anneal to complement through ________
hybridization
the complement and hybridization of DNA probe is labeled with ________
Marker
the complement and hybridization of DNA probe is labeled with ________
Marker
Colony blotting is used to identify which which ________ contain a sequence of interest
Clones
What is fluorescent in situ hybridization
fluorescently labeled probe is used to detect nucleotide sequences in intact cells fixed to microscope slides
In FISH, cells are visualized using ________ microscopy
Fluorescence
in FISH, probe binds to ________ sequences present in high copy number
RNA
