T4M3- DNA rep and mitosis

Question 1

what is PCR and who developed it

Answer 1

polymerase chain reaction developed by kary mullis in 1985
- allows us to copy or amplify DNA from small samples

Question 2

what is in the buffer of PCR

Answer 2

ions, salts and DNA primers

Question 3

what is role of dNTP

Answer 3

deoxyribonucleotide triphosphates act as building block for new DNA strands

Question 4

which machines are tubes placed in during PCR

Answer 4

tubocycular machines that goes through heating and cooling for DNA rep

Question 5

what is used to polymerize daughter cells in PCR

Answer 5

special DNA polymerase

Question 6

what 3 stages are included in replication during PCR

Answer 6

-denaturation
-annealing
-extension

Question 7

How is DNA heated and unwound

Answer 7

done with high temp- reaction heated to separate DNA strands

Question 8

what cools the solution and why

Answer 8

thermocycler cools sol’n to allow primers to anneal to complementary sequences

Question 9

what happens in extension phase of PCR

Answer 9

heat stable DNA polymerase extends and polymerizes daughter strands in 5’ to 3’ direction

Question 10

formula for number of copies

Answer 10

2^n

Question 11

what is gel electropheresis

Answer 11

used to separate DNA fragments from other sources

Question 12

where is DNA loaded in gel electropheresis

Answer 12

wells of poris gel

Question 13

where is the DNA and RNA attracted and why

Answer 13

attracted to pores because DNA/RNA is negatively charged and pores are positively charged

Question 14

how far do smaller molecules travel in gel electropheresis?

Answer 14

far

Question 15

who developed DNA sequencing and what was its purpose

Answer 15

Fredrick Sanger in 1975 and it was used to determine the sequence of DNA molecule

Question 16

what was the limitation of DNA sequencing

Answer 16

only determined the sequences of small fragments of DNA

Question 17

who discovered shotgun sequencing and what was the purpose

Answer 17

Gene Myers and Jim Webber- based on being able to break entire genome into different sized pieces and proceeding with 3 specific phases

Question 18

describe the first phase of shotgun sequencing

Answer 18

random sequencing of DNA in each fragment

Question 19

describe the second phase of shotgun sequencing

Answer 19

identifying regions of overlap and inferring long continuous sequence of nucleotide that makes up each chromosome

Question 20

describe the third phase of shotgun sequencing

Answer 20

annotating sequences to best identify regions of genomic DNA that encode genes, regulatory regions and non coding regions of DNA

Question 21

what is another name for sanger sequencing

Answer 21

dideoxy sequencing

Question 22

what is special about the deoxyribonucleotides in sanger sequencing

Answer 22

they are modified- do not contain a OH and the 3’ end

Question 23

what is the basis of dideoxy chain termination

Answer 23

the missing OH on the 3’ of ddNTPs does not allow for growing DNA strand

Question 24

what type of process is the insertion of ddNTps

Answer 24

random

Question 25

why is it important to fluorescently label each terminator

Answer 25

essential to be able to identify molecules where chain termination occured

Question 26

what happens to labelled strands after DNA synthesis and chain termination

Answer 26

loaded onto gel and fragments separated by gel electropheresis

Question 27

when does electropheresis continue until

Answer 27

each DNA band emerges from bottom of gel and laser excites dye attached to each deoxynucleotide

Question 28

what does the fluorescent detector record

Answer 28

amount emitted and mathed to one of 4 wavelengths attached to ddNTPs

Question 29

how is the assembly of dideoxy sequencing done

Answer 29

by complex algorithims in automated fashion using tech

Question 30

what is the assembly of final DNA sequence based on in shotgun seq

Answer 30

overlap of sequence similarities between fragments

Question 31

describe annotation

Answer 31

once DNA sequences are assembled, researchers annotate sequence and identify specific regions of interest along DNA

Question 32

how many reading frames are in DNA

Answer 32

6

Question 33

what does genome annotation require

Answer 33

identification of patterns or sequence motifs

Question 34

how can protein coding regions be inferred

Answer 34

based on identification of open reading frames, DNA or RNA sequence

Question 35

what do open reading frames contain

Answer 35

triplets of nucleotides

Question 36

what percent of eukaryotic genome repeated sequences that do not code for specific products

Answer 36

50%

Question 37

what do 50% of the eukaryotic genes code for

Answer 37

single copy genes, non coding RNA and repeated non coding sequences