Exam 3 Chapter 10 Flashcards
PCR initial denaturation
94-95* for 10-15 minutes
PCR cycles temperature
~55*
PCR final extension
~72* for 10 minutes
PCR hold
~4*
acts as a fridge for DNA
PCR cycles - denaturation
95* for 1 min
PCR cycles - annealing
attach primers forward and reverse
temp will vary - 55-65*
PCR cycles - extension
72*
number of cycles in PCR
20-50
over 50 cycles will use too much reagent
products of each cycle are used to
serve as the DNA template for the next cycle
each cycle ____ the amount of DNA synthesized in the previous cycle
doubles
RT-PCR
reverse transcription polymerase chain reaction
involves isolation of mRNA from a cell, using reverse transcriptase to convert it to cDNA, then amplification (cloning) of the DNA via PCR
then gel electrophoreses
uses of RT-PCR
detect and quantify RNA
study gene expression - compare it in different cells
1-step RT-PCR
all reagents go in one tube
reverse transcriptase added (enzyme)
add buffers, water, primers, polymerase, DNTPS
RT-PCR protocol
reverse transcription using RT enzyme
initial PCR activation
cycles
final extension
hold
RT-PCR - reverse transcriptase using RT enzyme
50* C for 30 min
RT-PCR - initial PCR activation
95* for 15 min
number of cycles for RT-PCR
35-50
RT-PCR - final extension
72* for 10 min is most common, but can involve trial and error
RT-PCR - hold temp
4*
limitation of RT-PCR
qualitative, not quantitative
don’t know by what factor genes are upregulated
SCVs
small colony variants
produced by some, not all, bacteria
auxotroph
mutant form
ex. lipoic acid -> cofactor for acetyl CoA to enter
lip A mutant -> no acetyl CoA
smiley effect
comes from the ladder of molecular markers sliding over to the side - forming a “smiley”
sometimes skip MM as a result
qRT-PCR
allow level of gene upregulation to be quantified
used in published research for more precision
uses a fluorescent probe
can qRT-PCR determine whether a person has had a virus in the past
no
a cell with greater levels of gene expression will be apparent in gel electrophoresis because
it will have a darker band
may indicate a greater need for the gene due to environmental change or stress
DNA sequencing
determining sequence of nucleotides within a long DNA molecule
DNA sequencing is helpful for
comparing DNA between organisms to show how they are related
sequencing proteins
aligning DNA/proteins to find differences (ex. insulin in a healthy person vs diabetic)
finding functional groups (domains) in proteins
Frederick Sanger
in 1977, discovered Sanger sequencing aka chain termination method
for determining nucleotide sequence of DNA
requirements for Sanger or dideoxy sequencing method
ssDNA template
primer
polymerase
one set of normal dNTPs
one set of ddNTPs
dNTPs
deoxynucleotides (dATP, dTTP, dCTP, dGTP)
have a 3’-OH required for chain elongation, to form phosphodiester bonds
ddNTPs
dideoxynucleotides (ddATP, ddTTP, ddCTP, ddGTP)
are randomly incorporated into chain
have no 3’OH so the chain ends when they are incorporated
Sanger methodology
ddNTPs randomly added during synthesis means that many fragments will be created when synthesizing the new strand based on the template strand
the ddNTPs that end each fragment will show up on the gel
can put together the entire strand by looking at the size of each fragment (big to small) and which ddNTP they end in
then find the actual sequence of the og strand
