Recombinant Technology
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What is the role of restriction enzymes?
They cut DNA in to manageable sizes called restriction fragments
Restriction enzymes usually:
1) Act as dimers and recognise short palindromic DNA sequences 2) Have precise recognition sequences 3) Some leave overhangs but others cut the DNA flush in the middle and are called blunt restriction enzymes
How can fragments be separated?
Gel electrophoresis
Which elctrode is DNA attracted to and why?
Positive as it has a negative charge
What is ethidium bromide used for?
To stain the DNA
What is ligase?
An enzyme which joins DNA fragments to create recombinant DNA
Cohesive termi are also known as what?
Sticky ends - this is because they can hybridize
How is recombinant DNA made?
Ligase is used to connect two DNA fragments
What are plasmids?
Small, circular, extra chromosomal DNA that occurs naturally in bacteria Have they’re own origin of replication Usually carry antibiotic resistance genes
How many kilobases does a plasmid hold?
30 kilobases
What is transformation?
Involves mixing bacteria with the plasmid DNA and creating temporary holes in the cell membrane by electroporation
What are competent bacteria?
Bacteria ready to take up new DNA
How are transformed bacteria selected for?
By using antiobiotic plates ie testing for antibiotic resistance
Where do we get the original DNA?
We make a library of genomic clones containing everything OR a library of cDNA clones
What is the advantage of genomic libraries?
Contains all the regulatory sequences allowing one to study transcriptional regulation
What is the advantage of cDNA libraries?
Way to identify which genes are expressed in a disease tissue compared to a normal tissue
What is the transcriptome?
Genes that are expressed in the original tissue
To isolate single clones from the mixed population it is important that there is only:
1) One insert into each plasmid 2) One plasmid in each bacteria 3) One bacteria starts with a colony
What are ESTs?
Expressed sequence tags
How are genomic libraries made?
You purify and digest chromosomal DNA with restriction enzymes
How is sequencing done?
1) Denature the template so it is single stranded 2) Cool with the primer 3) Start DNA synthesis reaction with DNA polymerase and dNTPs
What is the role of ddNTPS? (dideoxynucleotides)
Once they are added DNA polymerase can no longer extend
What is automated sequencing?
Up to 1000 nucleotides can be read in one reaction ie ddNTPs are labelled with a fluorescent dye which can be read allowing us to do a single reaction
What is done if you want to sequence DNA larger than 1kb?
Progressive sequencing