Recombinant DNA technology Flashcards
(20 cards)
What is meant by recombinant DNA
DNA that has been artificially formed by combining two or more fragments from different sources
What is the purpose of type II restriction enzymes
recognise and cleave at specific DNA sequences
- 4 or 6 bp in length
- sticky or blunt ends
- cutting sites are palindromic
- digest DNA to leave a 3’OH and a 5’-phosphate
What is the difference between EcoRI and Haelll?
EcoRI –> recognises 6bp target site - leaves sticky ends
Haelll –> recognises 4bp target site - leaves blunt ends
What is the purpose of DNA ligase?
forms covalent bonds between two DNA fragments with same sticky end
List the features of plasmids
- one or more unique restriction enzyme sites
- an origin of replication specific to host organism
- a selectable marker
Whats the difference between isoschizomers and neoschizomers?
isoschizomers - recognise same site and cut in same way
neoschizomers - recognise same site but cut differently
What are gene libraries?
large collections of cloned DNA fragments
What is the difference between genomic clones and cDNA clones?
genomic clones - cloning of chromosomal DNA –> used if promoter of intron-exon structure is to be analysed
cDNA clones - cloning of cDNA (DNA complementary to mRNA) –> used if protein is to be produced by recombinant bacteria
Describe how cDNA is made
- mRNA –> oligo-dT primer and reverse transcriptase
- mRNA + cDNA —> RNAase H + DNA polymerase I
- double-stranded cDNA
What are the requirements of PCR
- target DNA
- primers
- taq DNA polymerase
- dNTPs
- buffer containing Mg2+
- thermal cylinder
what is the difference between the forward and reverse primer?
forward - complementary to the 5’ end of the top strand
reverse - complementary to the 3’ end of the top strand
Describe the PCR cycle
- denaturation (95 C) - denature DNA into single strands
- annealing (40-65 C) allows primers to anneal to ssDNA
- extension (72 C) DNA synthesis
Describe the process of gel electrophoresis
- DNA an mRNA migrates towards positive electrode
- rate of migration of linear molecules is inversely proportional to the log10 of their size
- smaller fragments migrate faster
What dye is used to visualise DNA with UV
- ethidium bromide
- SYBR safe
Describe the process of cloning for protein expression
- select transformants
- isolate recombinants and check sequence
- grow large volumes
- induce expression from promoter
- pellet and lyse cells
- isolate and purify protein
Describe the process of making a vaccine
- insert gene encoding virus spike protein into suitable plasmid vector
- transcribe in vitro with uridine replaced by pseudouridine
- add 5’ cap and 3’ poly A tail to RNA
- remove vector DNA, purify spike protein mRNA
- package pure spike protein mRNA into lipid nanoparticles
Describe the different blotting methods
southern blotting –> DNA
northern blotting –> RNA
western blotting –> proteins
What is the difference between sanger sequencing and next generation sequencing
sanger sequencing - sequences a single gene or fragment
next generation sequencing - sequence whole genome or millions of fragments
Describe the process of making a transgenic plant using the Ti plasmid
- insert exogenous DNA from the Ti plasmid into T-region
- transform into agrobacterium
- infect plant cells
- exogenous DNA inserted into plant chromosome
- regenerate plant from manipulated cells
What is CRISPR?
- DNA sequences found in genomes of prokaryotes
- Cas = endonuclease enzyme that uses CRISPR sequences to recognise and cleave DNA
- isolated from streptococcus pyogenes
- re-engineered Cas9 to enable genome editing
