L7 - molecular bio techniques - DNA cloning & vectors Flashcards
what does the ability to isolate and manipulate DNA allow us to do? (6)
- isolate & modify & understand genes
- identify gene seq of mutant phenotype
- manufacture proteins & vaccines
- create transgenic organisms
- diagnose genetic diseases
- develop gene therapy
what is recombinant DNA technology
isolating regions of DNA and copying them
describe process of Recombinant DNA technology
- restriction enzymes cut DNA into fragments
2. copy DNA (eg cloning vectors - bacteria - self rep)
example of cloning vector
plasmid
how do R enzymes work?
recognise specific sequences and cuts both strands of sugar phosphate backbone
what part of the DNA do REs cut
sugar phosphate backbone
what are sticky ends?
after the cutting of DNA by REs a small region of single stand may be exposed
bases can easily be added here
what are blunt ends
cleavage of DNA where no single strand is exposed
importance of REs in bacteria?
cut up virus DNA as protection
how do bacteria prevent REs cutting up their own DNA
have methylase which adds methyl group to restriction site of its DNA to prevent RE cleavage
how many pieces does ECOR1 cut human genome
730,000
what is restriction mapping
creating a map of the positions that REs will cut DNA
how is restriction map made
- DNA cut with individual RE(first)
- separate DNA by size via gel electrophoresis
- compare each restriction fragments distance moved to markers
- pairs of RE then used to identify their relative sites
- repeat many times to make map
what does restriction mapping tell us
how many times an RE cuts DNA
the position they cut the DNA
what are restriction fragment length polymorphisms (RFLPs) and why are they useful
DNA sequence differs between individuals.
if a sequence differs in the restriction site the sizes and amount of restriction fragments will differ
allows differentiation between individuals - forensics etc
features of plasmids that make them useful cloning vectors
- contain ori which means they can replicate independent of chromosome
- contain antibiotic resistance genes to allow selective growth of bacteria with plasmid
- fast replication
describe process of producing coning plasmid
- cut plasmid and DNA fragment with same RE so they have the same sticky ends
- add the two together with DNA ligase
- DNA ligase reforms sugar phos backbone
- forms recombinant DNA plasmid which can be inserted into bacteria
what is multiple cloning sequence?
stretches of DNA put into the plasmid that can be recognised by many REs
why is multiple cloning sequence useful
plasmid can be opened by many REs so many options to match RE needed to cut DNA
why is a method needed to identify recombinant plasmids
the plasmid can just open and close again so recombinant ones need to be identified
describe method to identify recombinant plasmid
- MCS placed in lacZ gene
- lacZ gene codes B galactosidase which acts on X-gal to produce blue bacteria
- inserted recombinant DNA will disrupt gene
- blue colonies dont contain recomb DNA
white colonies do contain recomb DNA
list uses of cloned DNA
- create genomic library
- see organisation of genome
- identify changes in genome associated with phenotype/disease
- genetically engineer organisms
what is the genomic library?
huge library of all different DNA fragments of genome kept in plasmids
list other cloning vectors apart from plasmids
- bacteriophage vectors
- cosmids
- artificial chromosomes
what are bacteriophage vectors (and how big of a fragment can they hold)
vectors based on viruses that infect bacteria
25-30 kb
what are cosmid vectors
and how big of a fragment can they hold
modified plasmids
30-40 kb
what are artificial chromosome vectors
and how big of a fragment can they hold
plasmids that can clone large DNA fragments
can only produce a few copies
100kb - 2mb
how big of a fragment can plasmids contain
20kb
why can eukaryotic DNA not be cloned? what is done instead
because it contains introns which are large
mRNA is cloned as it contains no introns and then converted back to DNA (cDNA)
what is cDNA
complimentary DNA
complimentary to isolated mRNA
how is mRNA converted to cDNA
via reverse transcriptase which makes single complimentary strand
what are cDNA libraries
complimentary DNA copies of the mRNA (and therefore genes) expressed only in tissue the mRNA was extracted from
describe process of cDNA production
- mRNA isolated
- reverse transcriptase complimentary base pairs DNA to mRNA
- mRNA digested with RNaseH to separate strands
- complimentary base pairing to single stranded DNA (DNA pol & DNA ligase)
- double stranded DNA ligated into cloning vector
what is difference between cDNA library and genomic DNA library
- genomic DNA library - fragments of entire genome
- cDNA library - only copies of mRNA present in certain tissues (eg kidney cDNA library, liver cDNA library)
what are expression plasmids
cloning vectors that allow gene expression in bacteria
contain genes that direct T&T
used to produce large amounts of protein eg insulin
what are eukaryotic expression vectors
vectors that allow expression of cloned genes in eukaryotes
contain genes that direct T&T
define transformation
process of plasmid being taken up into cells
why may not all replicated plasmids contain the desired insert?
plasmid could religate with itself instead of insert
