Gene Technology Flashcards
What does recombinant DNA technology entail?
Using bacteria
Isolation of desired gene Insertion into vector Transformation into host Identification with gene markers Growth + cloning
Why can DNA fragments be inserted into genome of other organisms?
Genetic code universal
Mechanisms involved in translation + transcription = universal
What are the methods for gene identification?
mRNA to cDNA using reverse transcriptase
Describe viral replication
HIV binds to T cells Capsid fuses with membrane Reverse transcriptase converts RNA to DNA DNA moves into nucleus to create mRNA New viral proteins made HIV breaks away from T cell
Describe what happens in producing DNA fragments
Protein producing cell with large amounts of MRNA selected
Desired gene’s mRNA extracted
Reverse transcriptase forms single stranded complementary copy of DNA (cDNA) from mRNA
Hydrolysis of mRNA isolates cDNA
DNA polymerase forms DNA on template cDNA
Desired gene formed
Could be inserted into plasmids
What is restriction endonuclease?
Restriction enzyme
What does restriction endonuclease do?
Cuts DNA at specific sequence of bases
Producing either “blunt ends” or “sticky ends”
Why are sticky ends useful?
Are a palindrome Same forward + backwards 4 unpaired bases opposite Complementary plasmid Recognition sequence is 6bp palindromic sequence
Explain why pieces of human DNA would be able to join to the cut DNA of plasmids
Sticky ends
Complementary base pairing
Cut plasmids + lengths of foreign DNA can join. What features of their ends allow them to join?
Sticky ends
Complementary base pairing
What is the gene machine?
Used to create gene required
Synthesis of DNA from scratch
Why is the gene machine useful?
No need for pre-existing DNA template
How is the gene machine used to produce DNA fragments?
DNA sequence designed + fed into computer
1st nucleotide fixed to support
Nucleotide added in correct order
Protecting groups added to ensure bases added at right place
Nucleotides added Computer designs oligonucleotides
Protecting groups removed + oligonucleotides joined
Double strands formed to replicate gene
Gene inserted into plasmid
Vector stores, replicates + transfers gene
New gene checked for mistakes
Bacteria carrying gene cloned
In vivo amplification once DNA fragment containing desired gene you need is isolated you need to…
Clone it
Produce enough for medical or commercial use
What are the two main methods?
Vivo amplification
In vivo = transferring into host cell using vector
In vitro = polymerase chain reaction
Describe outline of vivo amplification?
Isolation Insertion Transformation Identification Growth
What does isolation + insertion require in vivo amplification?
Restriction endonuclease
Complementary sticky ends
Vector
Plasmid
Describe isolation + insertion
Plasmid cut open at desired gene recognition site using same restriction endonuclease
DNA fragment + plasmid sticky ends are complementary
Desired gene cut out using restriction endonuclease
DNA fragments + open plasmid mixed with DNA ligase
Plasmids contain new gene
Explain why the same restriction endonuclease enzyme must be used (4 marks)
Cuts same base sequence
Ensures sticky ends are complementary
Only cuts required gene
Ensures DNA fragments can be inserted into plasmid
For transcription to take place when preparing fragments what do you require?
RNA polymerase
Transcription factors to bind near gene
Promoter region = tell RNA polymerase where to bind
Terminator = releases RNA polymerase
What happens in transformation?
Vivo amplification
Introduce recombinant DNA into host
Mixing bacterial cell + plasmid in medium
Medium ice cold + contains Ca2+
Why is transformation in vivo amplification done in these conditions?
Ice cold + Ca2+
Ice cold chloride solution = increases permeability
Heat shock = encourages uptake
What are the three methods for identification in vivo amplification?
All use 2nd separate gene on plasmid that is
Resistant to antibiotic
OR
Make fluorescent protein
OR
Produce enzyme whose actions can be identified
