Molecular Diagnostics Flashcards
southern blotting
both probe and target nucleic acid are DNA
northern blotting
probe is single stranded DNA and target is mRNA
western blotting
target is protein or antibody
how does PCR work
dsDNA obtained from patient/individual/pathogen is denatured with heat and separated to ssDNA
primers flank each end of DNA and are allowed to anneal in cooler temps
deoxynucleotide triphosphates (dNTPS) are added
Taq polymerase synthesizes copy of DNA
Taq polymerase
synthesizes copy of DNA by extending the primers on both ends; DNA doubles in each cycle and becomes greatly amplified
advantages of PCR
very small amount of DNA is needed for the template; 10^9 fold increase from original DNA
disadvantages of PCR
you need to know the sequence of the flanking DNA for primer design
it’s error prone
you could amplify contaminated DNA
quantitative PCR (qPCR)
used to quantify copy number of a specific gene in two or more samples in real time
in addition to primers, it uses a probe which fluoresces only in presence of the PCR product
used for: detecting levels of an infectious agent and determining levels of gene expression
restriction fragment length polymorphism (RFLP) steps (5)
DNA fingerprinting; results is bands of DNA on nylon membrane
- DNA is cleaved by restriction endonucleases
- resolve DNA fragments on agarose gel
- transfer DNA to nylon membrane
- add radioactive DNA probe; wash off excess probe
- expose x-ray film to nylon membrane
how is sickle cell anemia shown on an RFLP
patients with sickle cell only have two restriction sites in their DNA, so their RFLP will only show one fragment on the membrane
normal beta globulin allele has 3 Ddel restriction sites and will show two fragments
variable number of tandem repeats (VNTR)
short tandem repeats (STR) occurs in normal genome and varies in individuals
VNTR repeat regions are isolated from genomic samples by flanking restriction sites or through PCR
useful in identification and severity of inherited diseases
ex: huntington disease, fragile X syndrome, frederich ataxia
how huntington’s is diagnosed
uses VNTR
PCR is done on healthy individual and patient with huntington’s; alpha-32 probe is attached to dCTP
resolve DNA fragments on urea-polyacrylamide gel
expose x-ray film to dried gel
huntingon’s will show much higher CAG segments
recombinant proteins
engineered to allow high levels of replication, transcription, and translation to occur in a host; cDNA of the protein is inserted into expression vectors
large scale production and purification
used to make insulin, growth hormones, erythropoietin, clotting factors, vaccines
how human insulin production works
insulin producing gene is isolated from pancreas cells
plasmid DNA is extracted from a bacterium and cut with restriction enzymes, forming plasmid vector
human insulin producing gene is inserted into the bacterial plasmid vector to form the recombinant DNA
how does Lispro improve insulin use
reverses the position of proline at position 28 with lysine at position 29 at C terminus of B chain
works in 2-4 hours instead of 6-8 hours
