recombinant DNA techh Flashcards
steps of setting up PCR (basic)
rna purification
then reverse trancription to get it to DNA
then PCR
> denature, anneal, DNA synthesis
which procedure needs a primer? A cDNA sythesis B genration of transgenic plants (Ti plasmid) C ligation of DNA fragment D restriction enzyme digestion E transcription in vitro
cDNA synthesis
> reverse transcriptase uses oligodT primer
> pairs with polyAAA tail and reverse transcriptase = hybrid
which enzyme is used to join 2 dna fragments with compatible sticky ends A rna polymerase B dna helicase C dna gyrase D Ddel E dna ligase
dna ligase!!
it ligates = joining by covalent bonds and can sythesise new phosphodiester bonds with ATP.
AquAdvantage Salmon are a transgenic animal that were made to produce more growth hormone. This was done by…
A Increasing their production of growth hormone stimulating hormone
B Adding the promoter for an antifreeze gene of another species
C Increasing the amount of somatotrophs present in the salmon’s anterior pituitary
D Adding a gene that prevents the decreases somatostatin release
OPTION B ..
AquAdvantage Salmon were made by by fusing the promoter for an antifreeze gene from an Ocean Pout, to the growth hormone cDNA from a salmoN
> ANTIFREEZE gene is present in all tissues so now growth hormone is present in all tissues instead of just pituitary gland
PCR is…
A The targeted amplification of a specific DNA sequence
B The amplification of a random DNA sequence
C The targeted amplification of a specific RNA sequence
D The amplification of a random RNA sequence
OPTION A
PCR is the targeted amplification of a specific DNA sequence by a billion fold. It is made specific by the synthesis of oligonucleotide single stranded DNA primers.
> does not work on RNA as taq polymerase doesnt recognise uracil
A transgenic animal is one that…
Has been cloned
Has exogenous DNA inserted into its genome
Has had a sequence of DNA removed from its genome
Has had its DNA undergo PCR
OPTION B
what is an advantage of using recombinant DNA tech in medicine - pregancy?
you can non inasively analyse foetal DNA
> also applications in foresnics using only a very small amount of blood
One stranded of a molecule of double stranded DNA has the sequence: 5ʹ-CGAA-3ʹ. What is the sequence of the complementary strand? 5ʹ-AAGC-3 5ʹ-ATCC-3ʹ 5ʹ-CCTA-3 5ʹ-GCTT-3 5ʹ-TTCG-3
OPTION E
as we go in the 5-3 direction
> dna is anti parallel
name some requirements for plasmids
UNIQUE single site for restriction enzymes
selectable marker that allows cells with plasmid to be distinguished from cells wthout it
how can we distinguish between recombinant plasmids and reformed plasmid (without the dna gene
the restriction enzyme site is in LacZ gene so recombinsnt plasmid cant make b-galactosidase so will not give a coloured dye.
how do the shine dalgamo and kozac sequences differ?
dalgamo pairs up with ribosome at binding site
kozac overlaps with initiation codon and needed for efficient translation
how can we overcome issues with expressing eukaryotic genes in E.coli?
use cDNA library/ mrna transcript which lack introns
use expression vectors that contain prokary promotoer, terminators and ribosomal binding site!
which enzyme is involved in PCR? A oligio-dT primer B dna ligae C rna polymerase D Taq DNA polymerase E Taq RNA polymerase
OPTION D
extracted from heat resistant bacteria so heat stable!
how does DNA profiling work?
uses short tandem repeat (2-6bp, 1-50 repeat)
> number of repeats vary in indiviuals so analyse lots of these STR
how is real-time PCR different to PCR
it measures the accumulation of amplified DNA after each cycle using a fluorescent probe
transgenic goat facts
we can get antithrombin (prevent blood clotting) from goats milk
> fuse the anththrombin gene with the milk protein gene
what ways can dna be damaged?
uv , gamma or x-rays
how can dna be repaired
homology directed repair - only in Sphase using sister chromatid as template
non-homologous end joining - any time but error prone as a INDEL is always added
which of the following is true of PCR? A denaturation at 72°C B primers 5-10bp in length C uses DNA polymerase from mouse cells D after 20 cycles, DNA amplified by 2^20 E synthesis at 55°C
OPTION D IS TRUE
denatuation 95, anneal 45, synthesis 72
dnapolymerase from thermophiles
primers are 20bp length!
Which of the following is NOT true of the polymerase chain reaction (PCR)?
A DNA denaturation 95 degrees
B DNA synthesis 72 degrees
C After 20 cycles of PCR the amount of the DNA
amplified increased 20x
D needs dATP dCTP dGTP dTTP
E needs primers complementary to ends of DNA
OPTION C
dna increase by 2 to power of 20!!
which of the following NOT needed for Sanger sequencing A DNA ligase B Dideoxynucleoside triphosphate C Deoxynucleoside triphosphate D A primer E DNA polymerase
OPTION A
Dideoxy is chain-terminating as it lacks a hydroxyl
> for a single gene/DNA fragment
which best describes reverse transcription
A mRNA as template synthesises complementry strand of DNA
B mRNA as template synthesises complementry strand of RNA
C protein as template synthesises mRNA encoding the protein
D mRNA as template synthesises mRNA encoding the protein
OPTION A
Using an mRNA molecule as a template, it synthesises a complementary strand of DNA
restrction enzymes can be obtained from A bacteria B fungi C algae D virus E plants
BACTERIA! restrction endonucleaese found in their immune system
how can we overcome expressing eukaryotic genes in bacterial cells?
use EXPRESSION VECTORS have
> prokary promotion and termination sequence
> ribosomal binding site
