L24- Molecular techniques Flashcards
why do we use restriction analysis?
- To investigate the size of DNA fragments e.g. small deletions
- To investigate mutations e.g. Sickle cell disease
- To investigate DNA variation e.g. DNA fingerprinting
what enzymes are used for restriction analysis
endonucleases
what produce endonucleases
bacteria
specific restriction endonucleases
recognise and cut specific DNA sequences (phosphodiester bonds) (at restriction sites)
restrictriction sites are mostly
palindromes
when restriction endonuclease cut at palindromic restriction sites we will ge an
uneven stagger
- producing sticky ends
- 5’ overhang- can be joined back together using ligases
restriction endonuclease are a type of
molecular scissors
Repertoire of restrictions endonucleases at our deplorable
e.g. BamHI, EcoRI, Pstl
how can restriction sites be predicted
using software
what alter restriction sites, meaning endonuclease will be useable to cleave DNSA
mutations
gene cloning uses
plasmids e.g. from E.coli
outline gene cloning
- Isolate relevant gene of interest following digestion with restriction enzymes
- Insert gene of interest into plasmid vector (recombinant DNA molecule)
- Introduce recombinant DNA molecule into suitable host cell e.g. E.coli
- Identify and isolate the clone containing the DNA of interest
plasmids
- Small circular dsDNA
- Found in bacteria
- Mini chromosomes
- Can transfer to other bacteria
- Often carry antibiotic resistance genes
why clone human genes
- Make useful proteins e.g. insulin (put into expression vector and gives us human insulin protein)
- To find out what genes do e.g. HTT
- Genetic screening e.g. Huntington’s, BRCA1/2, CF
- Gene therapy e.g. CF
polymerase chain reaction is a method of
amplifying small amounts of target sequences rapidly
requirements for PCR
- Thermostable DNA polymerase (Taq)
- Pair of primers (forward and reverse), uniquely defining the region to be copied
- Thermocycler
why use PCR?
- Amplify specific DNA fragment
- To investigate single base mutations e.g. Tay sachs and sickle cell
- To investigate small deletions or insertions
- To investigate variation, genetic relationships e.g. DNA profiling
process of PCR simple
- Denaturation
- Annealing stage
- Extending stage
denaturation
(95 degrees): DNA denatures (breaks H bonds and becomes ss)
annealing
(50-56 degrees) - specific primers added bind to complementary sequence
extending
(72 degrees): Taq polymerase added and DNA synthesis
PCR and copying of DNA is
exponential increase in DNA
primers are included in
the end product
DNA electrophoresis
DNA is negatively charged and will move towards the anode if placed in an electric field- DNA fragments can be separated on the basis of size (or shape).
