Download Biotechnologies Test: Restriction Endonucleases, Gel Electrophoresis, DNA Cloning, and PCR and more Exams Biochemistry in PDF only on Docsity!
BCHM 270 Biotechnologies Test
With Solution
Restriction endonucleases - ANSWER - found in bacteria naturally; these are enzymes that recognize and cleave double stranded dna at specific sequences
- this cleavage results in the production of dna fragments known as restriction fragments
why do bacteria use restriction endonucleases? - ANSWER these enzymes are used to recognize and identify foreign dna from invading species (e.g. viruses)
why do scientists use restriction endonucleases? - ANSWER used to clone dna
- combine dna sequences together
restruction endonucleas nomenclature: - ANSWER first: genus of bacterium
next two letters: species
number: order in which enzyme was discovered
e.g. HaeIII
gel electrophoresis purpose - ANSWER to separate dna segments based on charge
usually charge corresponds to size
method of gel electrophoresis - ANSWER separated on a gel (polyacrimde or agarose) + application of electrical field: the smaller the molecule, the farther it'll travel along the gel
dna cloning - purpose? - ANSWER to amplify a DNA sequence of interest
dna cloning - method - ANSWER 1. dna is cleaved using restriction endonucleases
- resulting fragments are joined to a dna vector, using dna ligase; this produces a hybrid molecule
- hybrid molecule is introduced into bacterial cell; bacterial cell divides, effectively producing a copy of the cloned dna
what are dna probes? - ANSWER - single stranded dna molecules that are labelled with a radioactive isotope or flurorescent dye
- dna probes carry nucleotides that are complementary to a target dna
specific polymorphism/mutation
describe the technique of ASO - ANSWER dna samples are mixed with ASO; aso will identify gene of interest; the probes will attach if dna sequence of interest is present and can be visualized
define blotting - ANSWER trasnfrer of macromolecules on a solid-phase membranous support
what are dna/rna molecules separated via gel electrophoresis blotted on? - ANSWER nylon or nitrocellulose membrane
purpose of southern blotting? - ANSWER to detect specfic dna sequences in dna samples; as well as the concentration/intensity of each dna fragement of interest
northern/southern blotting technique - outline. - ANSWER 1. gel electrophoresis - dna/rna is digested via restriction endonucleases and separated by size using gel electrophoresis
- separated fragements are trasnferred to a nylon/PVDF membrane + washed with blocking solution that woll reduce non-specific binding
- membrane is washed with a radiolabelled probe (or fluroscent dye) - probe will bind to dna sequences of interest
- target dna sequences are detected by the use of exposure film (radiolabelled probes) or fluroscent detectors; intensity of the bands produced is proportional to the concentration of the target dna sequence found,
PCR - purpose? - ANSWER to amplify a selected dna sequences; millions and billions of copies of a single sequence created in just a few hours
what components are required for PCR? - ANSWER DNA primer, dNTP nucleotides, Taq polymerase, magnesium, DNA sequence of interest.
what machine is used for PCR? - ANSWER thermocycler (can rapidly change temp.)
outline PCR method. - ANSWER 1. DNA sequence of interest, dNTP nucleotides, Taq polymerase, DNA primers are mixed in a tube
- thermocycler heats to ~95 deg. and denatures DNA into single strands
- reaction is cooled to ~55 def., and complementary primers anneal to strands
- temp. is increased to 65 deg. to limit non-specific DNA amplification. Taq polymerase adds free complementary dNTPs to DNA template; adds to the 3' end
- process is repeated several times
sequences on a chip.
dna microarray method - ANSWER normal and mutant gene are labelled with different fluorescent dyes and mixture is exposed to a small chip containing thousands of dna spots; each spot corresponds to a specific sequence
amount of fluorescence bound to a spot is a direct measure of how much dna is present in a sample.
What does CRISPR stand for - ANSWER Clustered Regularly Interspaced Short Palindromic Repeats
What is CRISPR? - ANSWER form of adaptive immunity in bacteria; these are sequences found within bacteria that can help protect them against invading species.
CRISPR method - ANSWER if a bacterial cell survives a virus; it will cleave the viral dna into small fragements, tagging the "good" dna and the "bad" dna - it will store this information in CRISPR loci (within the bacterial genome).
these fragments form the guide RNA sequences for Cas9.
how does the CRISPR loci help in protecting the bacterial cell? - ANSWER if cell comes into contact with viral dna; foreign sequences can be compared to the previously stored sequences; if they're a match, bacterial cell will destroy them using enzymes in the CRISPR/Cas9 system.
Cas9 stands for? - ANSWER CRISPR associated protein 9
what is cas-9? - ANSWER RNA-guided DNA endonuclease enzyme; associated with the CRISPR adaptive immune system.
How does cas-9 work? - ANSWER unwinds foreign dna and checks whether it has sequences complementary to the guide RNA, which has the previously stored CRISPR sequence.
If a match, Cas-9 will cleaves invading dna
