T4M3- DNA rep and mitosis Flashcards
what is PCR and who developed it
polymerase chain reaction developed by kary mullis in 1985
- allows us to copy or amplify DNA from small samples
what is in the buffer of PCR
ions, salts and DNA primers
what is role of dNTP
deoxyribonucleotide triphosphates act as building block for new DNA strands
which machines are tubes placed in during PCR
tubocycular machines that goes through heating and cooling for DNA rep
what is used to polymerize daughter cells in PCR
special DNA polymerase
what 3 stages are included in replication during PCR
-denaturation
-annealing
-extension
How is DNA heated and unwound
done with high temp- reaction heated to separate DNA strands
what cools the solution and why
thermocycler cools sol’n to allow primers to anneal to complementary sequences
what happens in extension phase of PCR
heat stable DNA polymerase extends and polymerizes daughter strands in 5’ to 3’ direction
formula for number of copies
2^n
what is gel electropheresis
used to separate DNA fragments from other sources
where is DNA loaded in gel electropheresis
wells of poris gel
where is the DNA and RNA attracted and why
attracted to pores because DNA/RNA is negatively charged and pores are positively charged
how far do smaller molecules travel in gel electropheresis?
far
who developed DNA sequencing and what was its purpose
Fredrick Sanger in 1975 and it was used to determine the sequence of DNA molecule
what was the limitation of DNA sequencing
only determined the sequences of small fragments of DNA
who discovered shotgun sequencing and what was the purpose
Gene Myers and Jim Webber- based on being able to break entire genome into different sized pieces and proceeding with 3 specific phases
describe the first phase of shotgun sequencing
random sequencing of DNA in each fragment
describe the second phase of shotgun sequencing
identifying regions of overlap and inferring long continuous sequence of nucleotide that makes up each chromosome
describe the third phase of shotgun sequencing
annotating sequences to best identify regions of genomic DNA that encode genes, regulatory regions and non coding regions of DNA
what is another name for sanger sequencing
dideoxy sequencing
what is special about the deoxyribonucleotides in sanger sequencing
they are modified- do not contain a OH and the 3’ end
what is the basis of dideoxy chain termination
the missing OH on the 3’ of ddNTPs does not allow for growing DNA strand
what type of process is the insertion of ddNTps
random
why is it important to fluorescently label each terminator
essential to be able to identify molecules where chain termination occured
what happens to labelled strands after DNA synthesis and chain termination
loaded onto gel and fragments separated by gel electropheresis
when does electropheresis continue until
each DNA band emerges from bottom of gel and laser excites dye attached to each deoxynucleotide
what does the fluorescent detector record
amount emitted and mathed to one of 4 wavelengths attached to ddNTPs
how is the assembly of dideoxy sequencing done
by complex algorithims in automated fashion using tech
what is the assembly of final DNA sequence based on in shotgun seq
overlap of sequence similarities between fragments
describe annotation
once DNA sequences are assembled, researchers annotate sequence and identify specific regions of interest along DNA
how many reading frames are in DNA
6
what does genome annotation require
identification of patterns or sequence motifs
how can protein coding regions be inferred
based on identification of open reading frames, DNA or RNA sequence
what do open reading frames contain
triplets of nucleotides
what percent of eukaryotic genome repeated sequences that do not code for specific products
50%
what do 50% of the eukaryotic genes code for
single copy genes, non coding RNA and repeated non coding sequences
