Genetic Lab techniques Flashcards
describe the three steps involved in the Polymerase Chain reaction.
Denaturation - DNA denatured
Annealing - cooling leads to DNA primers annealing to specific sections of the DNA to be amplified
Elongation - heat stable DNA polymerase replicates the DNA sequence following the primer.
What does a southern Blot measure
DNA that has been enzymatically cleaved and electrophoresis onto a gel. This is moved to a filter denatured and exposed to a radiolabelled DNA probe - This binds and is visualised on Film.
What does a Northern Blot show
RNA that has been cleave and ran on a gel. Gives reflection of gene expression.
What does a Western Blot show?
Sample proteins are deperated on a gel and labelled with ELISA and visualised. This is used for HIV tests.
Southwestern Blot
Identifies DNA binding proteins.
ELISA
Used to identify specific antigens or antibodies in a blood sample. Uses labeled antibodies bound to enzymes that produce a signal. (Direct ELISA)
Or uses a second antibody that is bound to an enzyme that produces a signal
What is FISH?
Fluorescent DNA or RNA used to probe specific sites of gene of interest on a chromosome. Can identify locations of genes and visualise anomalies such as microdeletions.
What is Karyotyping
Metaphase chromosomes are stained and visualised, ordered and counted to diagnose any chromosomal abnormalities such as Trisomies or sex chromosome disorders.
What is recombinant DNA
Insertion of a gene into a Plasmid vector containing antibiotic resistance genes. Can be used for generation of genomic libraries or make recombinant proteins for gene therapy.
What is a restriction Endonuclease?
Recognises palindrome sequences in dsDNA and cuts leaving sticky ends. used to make restriction maps of DNA, produce fragments.
What does reverse transcritase do?
Makes a complementary DNA (lacking introns) strand from mRNA. used to make recombinant DNA or in reteroviruses like HIV.
What are some methods of Modifying Gene Expression?
Transgenic strategies involve Random gene insertion into mouse genome.
Targeted Insertion or deletion of a gene through recombination with a gene.
What is the Cre-lox system
Inducibly manipulate genes to specific developmental points - study a gene whose deletion induces embryonic death.
What is RNA interference
dsRNA is synthesised that is complementary to the mRNA of interest. This is transinfected into human cells leading to dsRNA seperating and binding to the mRNA leading to reduced gene expression —> Knock out.
