lab 5 Flashcards
what areas did PCR have an impact on
- gene mapping
- cloning
- DNA sequencing
- gene detection
PCR template
any form of ds DNA
PCR ingredients
- reaction buffer
- DNA subunits
- DNA polymerase
- 2 DNA primers
- template strand
what 2 basic processes does PCR make use of
- complementary DNA strand hybridization
- DNA strand synthesis via DNA polymerase
DNA polymerase used
Taq polymerase from Thermus aquaticus
steps of PCR
- denaturation
- primer annealing
- primer extension
term for PCR machine
thermal cycler
temperature/thermal cycling
rapid heating and cooling of thermal block in thermal cycler
temp at denaturation step
94 degrees Celsius
temp at annealing step
60 degrees Celsius
temp at extension step
72 degrees Celsius
temperature cycle =
denaturation + annealing + extension
PCR generates DNA of a…
precise length and sequence
which cycle are fragments of precise length generated
third
what are targets of random insertions?
introns
what does SINE stand for
short repetitive interspersed elements
ex. of SINE
Alu element
where does Alu name come from
Alu I restriction enzyme recognition site found in this sequence
where are we looking for Alu sequence
PV92 locus on chromosome 16
dimorphic
Alu element present in some ind. but not others
homozygous Alu presence
941 bp
homozygous Alu absence
641 bp
heterozygous Alu
both 641 and 941 bp
bioinformatics
discipline that integrates mathematical, statistical, and computer tools to collect/process biological data
InstaGene matrix
consists of suspension of negatively charged beads which bind divalent cations
- need to remove divalent cations because they will assist enzymes in DNA degradation
supernatant
contains clean, intact genomic DNA
functions of incubation steps
- breakup of connective tissue
- inactivates DNases
master mix
mixture of nucleotides, buffer, and Taq DNA pol
dye that makes DNA primers yellow
tartrazine
is manual PCR possible?
yes
