Manipulation of DNA Flashcards
What are restriction enzymes?
They cut DNA
They recognise specific nucleotide sequences and cut off both strands
They leave a blunt end known as a sticky end
How to clone DNA?
DNA is cloned by inserting into a plasmid which allows for replication
DNA and plasmids are then cut with the same restriction enzyme so they have complementary adhesive ends
Cut DNA and plasmid fragments are mixed and anneal together
DNA ligase will ligate the DNA and plasmid fragment together to form recombinant DNA molecules
E.coli can be used to take up individual plasmids and replicate them
What does DNA replication allow for?
Mapping and sequencing of the genome Identify genome changes Genetically engineer organisms Study and production of therapeutics Identifying differing expressed genes Characteristic genome organisation Gene therapy
How to express eukaryotic proteins?
You need DNA copies of transcribed mRNA as it doesn’t contain introns
cDNA is a copy of mRNA produced using reverse transcriptase
The use of Oligo primers?
Oligo primer is added to mRNA, then reverse transcriptase so the DNA synthesis is continuous
RNA us partially digested with RNase H
DNA pol 1 and DNA ligase seal gap creating two double stranded cDNA which can be used with vectors in E.coli
How to stop DNA polymerase?
Dideoxy nucleotides interrupt DNA polymerase in dideoxynucleotide chain termination sequencing
What are Deoxynucleotides?
Incorporated by the formation of phosphodiester bonds between the 5’ PO4 group of nucleotides being incorporated and the 3’ OH of the previous nucleotides
How Dideoxynucleotides stop DNA replication?
They dont have a OH or 3’ carbon and so DNA cannot incorporate further nucleotides after Dideoxynucleotides are incorporated as they have no 3’ OH
Uses of the PCR?
Make copies of DNA without a vector
Can amplify fragments from small amounts of DNA
The 3 stages of PCR?
1) Denaturation - denature DNA to single strands
2) Primer annealing - allows primers to hydrolyse their complementary target DNA sequences
3) Primer extension - TAQ polymerase can synthesis DNA
What separates DNA fragments after PCR?
Gel electrophoresis
What are DNA microarrays?
They use nucleic acid hybridisation to rapidly measure expressed genes
