Flashcards in molecular genetic technique and applications Deck (22):
In what 2 situations is molecular genetics used
you know the identity of the mutation.
you don't know the identity of the mutation.
what does PCR detect.
presence or absence of a product.
detects the size of the product.
How is PCR carried out
Heat denaturation 94 degrees Celsius- breaks hydrogen bonds but not phosphdiestersase bonds.
Primer annealing 55 degree Celsius.
Primer extension 72 degress Celsius.
Exponential growth of the DNA.
what molecule are the primers in PCR
what method is used to separate PCR products
what charge does DNA have
The DNA is always negatively charged due to the phosphate group on the phophdiesterase bonds.
In gel electrophoresis which molecules move the furthest
Uses of PCR.
determine the presence or absence of a product- allele specific.
determine the product size by gel electrophoresis
which 2 conditions are example where PCR can be used to determine their diagnosis
cystic fibrosis- 3bp deletion (altered product size).
Connexion- single bp deletion causes deafness.
uses of oligonucelotide ligation assay
Allele specific mutation detection- uses when you know the identity of the mutation (point mutation)
How is oligonucelotide ligation assay carried out
2 Phases -PCR and OLA
Primer is hybridized to the target sequence. The primers are designed with either the normal or mutant nucleotide(s) at the 3' end and a tail of different lengths to distinguish various PCR products based on size at the 5' end. A PCR reaction is performed.
A common primer contains a fluorescent dye marker (FAM, TET, HEX) at the 3' end and meets the first primer right over the nucleotide position that will be altered in a mutant allele. If the 3' end of the first primer matches perfectly with the target DNA, it will be brought into close enough proximity to the second oligonucleotide that both primers can be ligated together. No ligation occurs when there is a mismatch between the 3' end of the first primer and the target DNA.
what are the main disadvantages of mutation analysis.
cannot detect a mutation that it isn't designed for
gene might be to big for PCR.
GC-rich regions are difficult to PCR!- because there are 3 hydrogen bonds.
how is southern blotting carried out.
place DNA on argose gel and separate by gel electrophoresis.
Blot DNA fragments from argose gel onto membrane.
Membrane is imprinted with DNA bands.
Add a labelled probe to the membrane.
detection reveals a band where your probe bound to the target sequence.
what condition is southern blotting used to detect
Fragile X syndrome.
When is DNA sequencing used
when you don't know what the mutation is.
detect changes in sequence of the gene.
characterize the nature or position of a novel mutation.
how does the chain termination (sanger) method work.
• starting material =single DNA fragment (produced by PCR)
• anneal a specific oligonucleotide primer and synthesize a new complementary strand
• strand synthesis catalysed by a DNA polymerase, and requires deoxynucleotides (dATP, dCTP, dGTP and dTTP) to form the new strand
• BUT a small amount of each dideoxynucleotide is also incorporated (ddTTP etc.)
– the DNA pol does not discriminate between dNTPs and ddNTP
– ddNTP is incorporated into the growing DNA strand
– BUT ddNTP lacks the 3’hydroxyl group form a phosphodiester bond with the next nucleotide phosphate
what are the results from Sanger method
• new strands are synthesized, all different in length, and each ending in a ddTTP
• this happens every time that a dATP occurs in the original template fragment
• other chains will be terminated by ddATP, ddCTP and ddGTP
• termination will occur randomly
• separate by size via gel electrophoresis.
For what conditions is the Sanger methods used to distinguish between different muscular dystrophies.
MEGF10 mutation screening- distinguish between different muscular dystrophies that are clinically identical
• DNA sequencing enables you to sequence the patients DNA and therefore the point of mutation by comparing it to the normal DNA sequence (because you know the full DNA sequence including exons and introns)
what are the 2 types of DNA sequencing
what forms of DNA use clonal sequencing
• “long” PCR product covering a part or the whole of a gene
• panels of selected genes known to be mutated for a particular phenotype
• very large “exome” panels, based on all coding genes in the human genome.
method of clonal sequencing
• captures genomic material of interest for clonal sequencing of selected genes getting specific genes from the genome to sequence them)- bait (labels them with oligos) and magnet are used to extract them so they can be clonally sequenced.