Flashcards in Use of Molecular Genetics in Medicine Deck (40)
what is a chromosomal disorder?
defect in whole chromosomes, not just single mistake
what are the solutions for doing genetic testing?
enzymes that manipulate DNA
cloning of target DNA into a vector
Polymerase chain reaction (PCR)
what is hybridization?
annealing of ssDNA probe to complementary ssDNA
what do PCR (primer), sequencing (primer), southern blots, RFLP analysis and allele specific oligonucleotide (ASO probes) use?
what does northern blots use?
what does western blots use?
what is restriction endonucleases (class II)?
recognize and cut DNA in a specific and reproducible fashion.
hydrolyze phosphate backbone to give a 5' phosphate and a 3' OH
blunt or sticky overhang 4-8 bp in length, 6 bp is most common
homodimers that recognize short, symmetric DNA sequences
Do not have methyl's activity and thus are very useful for DNA manipulation
what is a palindrome?
when read in the 5' to 3' direction, the sequence on the "top" strand is identical to that of the "bottom" strand
what is the probability of the restriction site occurring in a piece of DNA?
expect that a Hpall recognition site of 4 bp will occur every 256 bp
what is DNA polymerase?
enzymes that synthesize a complementary new strand of DNA from DNA or RNA template
what are the 4 most used types of DNA polymerase?
DNA polymerase I
Klenow fragment DNA polymerase
what is prokaryotic plasmids?
single, large circular chromosomes - can insert up to 10 kb of foreign DNA
what are 3 important feature of cloning vectors?
must have multiple cloning sites
must have a selectable marker
what are the functions of cloning vectors?
what are the founding priniciples of PCR?
DNA polymerase I synthesize in 5' to 3'
primers - free 3' OH
heat to denature DNA
exponential increase in the number of DNA molecules
what are the components required for PCR?
deoxynucleoside triphosphates (dNTPs)
what is a cofactor required for DNA polymerase activity?
what are the steps in PCR?
REPEAT 28-35 times!
in gel electrophoresis, what do the nucleic acids migrate towards and why?
towards the cathode, nucleic acids have a negative charge from phosphate backbone
what is the difference between northern blots and southern blots?
norther blots contain isloated mRNA separated by gel electrophoresis
why do we sometimes call plasmids vectors?
vectors carry DNA of interest
what do we need for easy primer design?
information from the sense strand - then we can make both forward and reverse primers from that
what is the forward primer complementary to?
what makes primer design so difficult?
you want to get primers that work together
anneal and melt at the same temperatures
depends on GC primers (3 hydrogen bonds)
how do we amplify mRNA?
we need ds species to PCR and clone
use reverse transcrpitase to take RNA and convert it to DNa
in gel electrophoresis, which molecules move faster and slower?
fast = small
slower = large
what was the first diagnostic technique devides?
what does southern blot tell us?
if one or both alleles are different from normal
if we have made or broken a restriction site
what does western blot tell us?
if there is a change in size of the protein of interest
use antibody specific to protein of interest
what do DNA and RNA techniques use?
what does protein technique use?
what did Freddy Sanger do and how?
developed the human genome project
took 3' OH off the ribose
what technique is commonly use today?
what occurs in sequencing?
one lane on a gel, products of each reaction will emit a different color when it excited by light
what can a single base pair change in RFLP analysis?
change the creation or destruction of a restriction site
destroys Sca1 restriction site
First: amplify 186 bp frag of MSUD gene
Next: digest with Sca1 and see on agarose gel
what is used to detect polymorphins or common genetic mutations (CFTR, sickle cells, others)?
ASO (allel specific oligonucleotide) probes
what can allele specific PCR detect?
single nucleotide changes
what ensures that the PCR is working properly?
what does sickle cell mutation of beta-globin gene destroy?