Recombinant DNA Techniques

Medical Significance of the use of recombinant DNA

Detection and Prediction of disease and disorders

Prevention and treatment

Personalized medicine

What is recombinant DNA

Combination of two or more DNA sources

How do we separate DNA or RNA molecules by size

Gel electrophoresis

Components of PCR

Thermostable DNA Polymerase, nucleotides, synthetic primers and DNA

How does each cycle of PCR begin

The melting of double stranded templates followed by the annealing of synthetic primers

By how much does each cycle duplicate the number of copies produced in previous cycle?

Doubles it

Real Time PCR (quantitative) is used to determine

The number of copies of specific sequences in a cell and compare the experession of genes in different cell types or tissues

What kind of probe does RT-qPCR use

fluorescent

What direction do nucleic acids migrate in gel electrophoresis

From negatively charged cathode to positively charged anode

How are fragments separated by size in gel electrophoresis?

shorter fragments move faster, longer fragments move slower

What is the purpose of hybridization

Detect sequence of interest using labeled probes

What technique must be used before Northern and Southern Blot

Gel electrophoresis

Diagnostic Applications of FISH

identification of numerical and structural chromosomal abnormalities, tracking origin of cells after bone marrow transplantation, identification of regions of deletion or amplification

What is the Philadelphia chromosome?

Specific abnormality of chromosome 22, which is unusually short and is most commonly associated with CML

What does AbI gene in Philadelphia chromosome express

Membrane associated tyrosine kinase

How does AbI gene cause unregulated cell division?

Translocation of AbI into chromosome 22 forms a fusion gene BCR-AbI. This fusion protein is “always on” or constitutively active

Most restriction endonucleases recognize

Palindromic sequences

Restriction Fragment Length Polymorphism (RFLP) allows what?

Detection of point mutations that destroy or crease restriction sites. Eg. sickle cell anemia

Detection of Variable Number of Tandem Repeats (VNTR) is preformed by

PCR amplification of the repetitive DNA, used in genetic testing, forensic, DNA fingerprinting

What allows screening of many gene expression simultaneously?

Microrrays

How does Sanger sequencing work?

Uses termination of DNA synthesis at specific nucleotides during DNA synthesis by DNA polymerase

How does Next Generation Sequencing work?

Uses DNA synthesis termination principle, allows simultaneous sequencing of millions of DNA fragments