Molecular Genetic Techniques 1 (L5) Flashcards
modes of inheritance
dominant, recessive, X-linked, mitochondrial
relationship b/w genotype and phenotype
genotype determines phenotype
two different modes of cell division
mitosis and meiosis
markers (biomarkers)
polymorphisms on DNA that are co-segregated w/ the disease phenotype, which presumably are located very close to the disease-causing gene
haplotype
combo of many polymorphisms along one chromosome
what is recombination efficiency dependent on?
distance from a polymorphism or mutation
what is the overall purpose of linkage analysis?
use landmarks that we know on a chromosome to determine where the disease-causing mutation is
alleles
different forms of the same gene
what is a technique to analyze markers on DNA?
agarose gel electrophoresis
agarose gel electrophoresis
agarose creates 3D sieve to analyze biomolecules - DNA travels through pores based on size (larger travel slower, smaller travel faster) -> incubate with fluorescent dye to visualize bands
restriction enzymes
proteins from bacteria that attack bacteriophages and chop them into smaller pieces
what do restriction enzymes recognize?
palindromic sequences in DNA
what type of cuts do restriction enzymes make?
sticky end cuts - leave some ssDNA out
blunt end cuts
RFLP
restriction fragment length polymorphism - mutation eliminates a restriction site -> change palindromic sequence -> restriction enzyme no longer recognizes it -> no cut
southern blot
- cleave DNA with restriction enzymes
- run on agarose gel
- transfer DNA to nitrocellulose membrane in alkaline solution
- stain by radioactive probe
- visualize bands
what does alkaline solution do to DNA?
denatures it
probe
antisense sequence w/ radioactivity or dye for visualization
microsatellites
short tandom repeats of DNA sequences - can have di-nt or tri-nt repeats
SNP
single point mutation
polymorphism vs. mutation
polymorphism: neutral, mostly doesn’t cause harm
mutation: affects body fxn, changes codon or how gene is expressed - can cause a disease state
what are three types of polymorphisms DNA can have?
- RFLP
- microsatellite
- SNP
genetic distance
how close a marker is to the disease gene - measured in centimorgans (cM) - can do this to calculate how far apart marker is from disease gene, then use probability fxn to determine how likely is it to find a disease gene at a particular location
what does 1 cM correspond to in bp?
1 million bp
relationship b/w genetic distance and chance of recombination
smaller the distance, smaller chance of recombination - if very closely linked, most often get parental genotype
what can you use to determine how much the disease gene is linked to the marker?
percents of non-recombinant and recombinant gametes
family-based linkage analysis
start with a family -> use polymorphic marker -> go through recombinations -> track where it goes
what are other applications of DNA polymerisms in medicine?
paternity tests and forensic analysis - criminal identification
how to pinpoint the mutated gene?
cytogenetic map -> linkage map -> physical map -> sequence map
genetic vs. physical map
genetic: chromosome banding pattern FISH
physical: hybridization to plasmid clones
sanger dideoxy sequencing
- Add a primer to the DNA
- Add DNA pol + dATP, dGTP, dCTP, dTTP
- Add ddNTP in low concentration
- ddNTP gets incorporated occasionally
- when ddNTP incorporated, DNA pol can’t extend further
- get multiple lengths depending on where the ddNTP binds
- repeat sequence with all four ddNTPs w/ fluorescent colors
- analyze w/ PAGE
why does a dideoxy nt stop DNA polymerase?
it has no 3’ OH, which is necessary to extend DNA
PAGE
polyacrylamide gel electrophoresis
-each band is one nt from last one
-each band has fluorescent color
read sequentially -> get DNA sequence
how can you use sequencing to find a mutation?
compare patient to control electropherogram - differences indicate mutation
PCR
cycles of denature, anneal, extend
what ingredients are necessary for PCR?
- target DNA
- DNA pol
- dNTPs
- MgCl2
- buffer
applications of PCR
- diagnosis
2. mutation analysis
what increase in DNA does 20 cycles of PCR correspond to?
1 million fold increase in DNA
