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Molecular 3rd pt. 2 > Lecture 1 > Flashcards

Lecture 1 Flashcards

1
Q

what was the first genome sequence composed of

A

many genome donors all together

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2
Q

when do we care about insects enough to sequence their genomes

A
  • model organisms
  • or ones that spread disease/ affect us
  • or have agriculture relevance (like bee pollinators)
  • pest control
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3
Q

why were the first dna sequencing machines so expensive

A
  • cause you had to develop the technology,
  • later ones were cheaper since you already had a reference
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4
Q

true/false the health and ancestry commercial dna analysis kits are for whole genome sequencing

A
  • false
  • theyre for genotyping
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5
Q

how do the health and ancestry dna analysis kits genotype

A
  • they use “gene chips” that detect single nucleotide polymorphisms (SNPs)
  • the more snps in common, the more related
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6
Q

true/false we can sequence DNA without breaking it up

A

false

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7
Q

briefly describe the shotgun strategy

A
  1. dna extraction
  2. dna fragmentation
  3. clone into vectors
  4. transform bacteria, grow and isolate vector dna
  5. sequence the library
  6. assemble contiguous fragments
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8
Q

how do we sequence the library in the shotgun strategy

A
  • randomly
  • we’ll figure out how they all relate to each other later on
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9
Q

what strategy requires assembly of reads into contigs

A

shotgun

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10
Q

what is a contig

A

a series of overlapping dna sequenced used to make a physical map that reconstructs the original dna sequence of a chromosome or a region of a chromosome

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11
Q

what strategy is often used to close the gaps in shotguun sequencing

A

“primer walking” strategy

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12
Q

what strategy is often used to obtain the sequence of a short region of DNA

A

“primer walking” strategy

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13
Q

if you only want to synthesize 1kB of DNA, what should you do

A

use “primer walking” strategy

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14
Q

true/false “primer walking” strategy is often used to sequence full genomes

A

false

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15
Q

briefly describe “primer walking” strategy

A
  1. start sequencing from specific site in genomic DNA or chromosome
  2. design primer at a site based on sequence info obtained
  3. start sequencing w newly designed primer
  4. repeat 2 and 3
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16
Q

describe the relationship between the “shotgun” and “primer walking” strategies

A
  • shotgun is done to get most of it
  • primer walking comes in to fill in the gaps
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17
Q

which is more orderly between “shotgun” and “primer walking” strategies

A

primer walking

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18
Q

true/false in primer walking you always know what came before it and what comes after

A

true

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19
Q

how do you decide primers for primer walking

A

as you go

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20
Q

sanger’s sequencing is based on what kind of synthesis f DNA

A

in vitro

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21
Q

true/false sangers sequencing is still frequently used today

A
  • false
  • has nasty chemicals and hard to scale up
  • hardly used
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22
Q

what would happen in sanger sequencing if too much ddA is present in the A sample

A

all the resultant DNA strands would be very short since the chain termination would occur very early in the reaction

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23
Q

what happens when modifed nucleotides are added during DNA synthesis in sanger sequencing

A

causes chain termination

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24
Q

describe sanger sequencing

A
  • you’ll have a pool of normal ATGC
  • and a tiny amount of the dideoxy (modified) ones
  • anneal a primer and add polymerase to add its studd
  • once the ddATP gets added, we know what base is there (cause if we only have modified As, then an A must have gone there)
  • these strands are separated by size via gel electrophoresis
  • then repeat with the other nucleotides to see the other bases
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25
Q

is DNA sequencing read from the bottom up or top down

A

bottom up

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26
Q

is the gel used for sanger sequencing the same as what we use in lab

A
  • no
  • this gel (polyacrylamide) can separate the strands by just one nucleotide
  • way more sensitive
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27
Q

how many primers are annealed to the DNA strand in sanger sequencing

A

one only

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28
Q

when cannot we design a primer in sanger sequencing

A

if no previous sequence info is known about the dna template

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29
Q

when do we need a new template in sanger sequencing

A

for every new DNA template to be sequenced

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30
Q

how will we know what the primer should be for the plasmid vector in sanger sequencing

A

we’ll know the entire sequence of the plasmid so we can make primers for whatever region we’re interested in

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31
Q

how do we go from the plasmid vector to the DNA of interest

A
  • cut the plasmid with RE X
  • insert DNA
  • denature plasmid DNA for sequencing by heating it up
  • anneal one primer to the region of interest (that we’ll know because we know the entire sequence of the plasmid)
  • split samples into 4 tubes and do the steps
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32
Q

why can’t we anneal more than 1 primer to the plasmid vector in sanger sequencing

A
  • because you won’t be able to identify which segments are from which plasmid
  • they might be the same length which will confuse you when you try to run the gel
  • overlapping bands
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33
Q

what are the 2 primers for the plasmid vector in sanger sequencing

A
  • M13 forward
  • M13 reverse
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34
Q

what would happen if you anneal forward and reverse primers at the same time in a single sample

A

you get sequences from both strands of the DNA template

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35
Q

why would you anneal forward and reverse primers at the same time in a single sample

A

can verify that they’re the reverse complement of each other, if they’re not… smth isn’t right

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36
Q

what is the problem w manual sanger sequencing

A
  • can only read 150-200 nucleotides per gel
  • its okay w short sequences but not big
  • very labour intensive and time consuming
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37
Q

what is the main difference in automated sanger sequencing over manual

A

they use a diff colour fluorescent dye to tag each ddNTP

38
Q

how many tubes are used in manual sanger sequencing

A

4

39
Q

how many tubes are used in automated sanger sequencing

A

1

40
Q

how does automated sanger sequencing work

A
  • when nucleotides are added to the chain, a fluoresence is released based on the ddNTP
  • the fluorescence detector relays its signals to the computer which interprets the colours to ddNTP
41
Q

what does it mean in the chromatograph for automated sanger sequencing when a peak has two colours

A

could be evidence of a heterozygote (one allele is G, the other A)

42
Q

what are the advantages of automated over manual sanger sequencing

A
  • Can read up to 900 nucleotides per Rx
  • Allows automated reading and recording of results
  • Cost effective
  • Can perform all 4 ddNTPs Rx in one sample and load in same lane of gel
  • Can sequence up to 384 diff DNA samples simultaneously by using gels formed within capillary tubes
43
Q

what is the most popular 2nd gen dna sequencing

A

sequencing by synthesis- SBS (Illumina)

44
Q

whatre the disadvantages of 2nd gen methods over sanger

A
  • geared towards large # of samples, highly impractical for small samples
  • requires high computing and data storage capacity
45
Q

in next gen sequencing, what can be eliminated

A
  • interting/ cloning the DNA into a vector
  • transformation of vector into bacteria
  • isolation of plasmid from transformed bacteria
46
Q

what is done instead of all the plasmid steps in next gen sequencing

A

ligating “adapter sequences” to each end of DNA fragment and PCR amplify

47
Q

how do you prep a genomic DNA library for next gen sequencing

A
  • isolate genomic dna
  • fragment genomic dna
  • ligate dna primers/ adapters/ tags to each end of genomic dna fragments
  • attach tagged dna fragments to slide
  • pcr amplify to obtain large # of molecules of the same fragment in clusters or spots on the slide
  • perform da sequencing reaction directly on the slide by passing diff solutions over slide
  • record signal after each slide
48
Q

why is a pcr step added in the prep of a genomic DNA library for next gen sequencing

A

we need more material to build up the signal

49
Q

why are adapters added in the prep of a genomic DNA library for next gen sequencing

A
  • you want to PCR, so you need the primer and if you add the adapters you can know the sequence of the adapters
  • also helps you anchor your dna samples onto a solid suuport
50
Q

true/false in illumina sequencing, only modified nucleotides are used

A

true

51
Q

what dna sequencing technique is done by synthesis, but without permanent chain termination

A

illumina

52
Q

where on the nucleotides in illumina is the fluorescence added

A

on the base

53
Q

where on the nucleotides in illumina is the block added

A

the sugar

54
Q

does illumina have multiplexing ability

A

yes, up to 50 million spots can be analyzed at the same time

55
Q

how many altered nucleotides are added for illumina sequencing

A

all 4 are added in each cycle

56
Q

what is the label for the 3 phosphates on the nucleotides

A
  • alpha (closest to the rest of nucleotide)
  • beta
  • gamma
57
Q

when the phosphates get cleaved (for the addition of the nucleotide) which one remains

A

alpha

58
Q

in each cycle of illumina, how many nucleotides are detected

A

one

59
Q

would a signal with 2x the average intensity be likely due to 2x incorporation of the same nucleotide in the same cycle in illumination sequencing?

A
  • no
  • the 3’ block stops more than one nucleotide from being added during a single cycle
  • this cannot be done
60
Q

the identify of each base of a cluster/ spot is read off what for illumina

A

read off sequential images/ photos taken after each sequencing cycle

61
Q

what are 2 3rd gen dna sequencing methods

A
  • single molecule real-time sequencing (SMRT)
  • nanopore sequencing
62
Q

what sequencing method doesn’t require any dna synthesis

A

nanopore sequencing

63
Q

what is one instance where you can bring a sequencing device to the scene

A

the small portable nanopore sequencing device (MinION)

64
Q

how does nanopore sequencing work

A
  • we monitor changes to an electrical current as a single-strand DNA moved through a tiny pore in a membrane
  • each nucleotide base causes a characteristic change to the current
65
Q

why do 3rd gen sequencing have easier genome/contig assembly

A

because they can do really long reads (its like do a 10 piece puzzle, rather than 1000 piece)

66
Q

what is the disadvantage of SMRT over illumina method

A
  • higher error rate
  • increased cost
  • not as many DNA sequencing centers have access to this technology
67
Q

what are 3rd gen very useful for

A
  • tracking outbreaks
  • used for covid to see what outbreaks are out there
68
Q

what is HiFi PacBio

A
  • like SMRT but you sequence many times, rather than just once
  • makes it easier to detect errors
69
Q

why is SMRT very fast

A
  • it is uninterrupted synthesis by a single DNA polymerase
  • no pcr amplification of template DNA is required
70
Q

how does SMRT work

A
  • we have a bunch of microwells, that have a single DNA polymerase attached to the bottom
  • the DNA template is gonna be sequenced, and is held by the polymerase
  • there are pools of dNTPs in the wells
  • each labeled w a diff colour fluorescent tag attached the the gamma phosphate
  • only the dNTP help by the polymerase as its about to be added will be dected by the laser/ detector at the bottom
  • they record the pulse of light coming from the fluorescence
  • when the dNTP is added, the phosphate (and fluorescence) is cleaved off, and the light is lost
  • the next dNTP is added and it continues
71
Q

what do the pulses of diff colour correspond to in SMRT

A

diff dNTPs being incorporated

72
Q

briefly describe this

A

yellow means C is being held, and therefore incorporated, blue is the A being held and incorporated after

73
Q

explain the diff peaks

A

no correlation to nucleotide being added
- very random

74
Q

what does the personal genome project do

A
  • sequenced the genomes of 100 000 people
  • to correlate genotypes w health, physical and ancestry info provided by participants
75
Q

what does the cancer genome project do

A
  • sequenced the dna from primary tumours and normal genomic dna from the same people for 1000 cancer patients
  • evaluated 350 human cancer cell lines and their response to 18 drugs to correlate drug sensitivity w specific genotypes
76
Q

what does the pediatric cancer genome project do

A

sequenced and compared normal and tumor tissue samples from 600 pediatric cancer patients to find genetic causes of childhood cancers

77
Q

true/false dna sequences for both strands of a double stranded dna template are usually obtained to check for any technical errors in the sequencing procedure

A

true

78
Q

how many potential protein sequences can be derived from a double-stranded dna fragment

A

6

79
Q

once you have obtained the sequnce of a DNA fragment, how do you locate the genes/ which of the reading frames is the right one

A

look for the longest “open” reading frame within DNA sequence

80
Q

what is an open reading frame

A

the sequence of DNA that encodes a
continuous stretch of AA before encountering a stop codon

81
Q

what are the 3 stop codon

A
  • UAA
  • UAG
  • UGA
82
Q

what amount of codons are stop codons

A

3/ 64 OR 1/21.3

83
Q

which reading frame is best

A

2

84
Q

which reading frame is best

A

3

85
Q

what do the green boxes mean

A

stop codons

86
Q

what do the red bars mean

A

continuous open reading frames

87
Q
A

1

88
Q

which reading frame is best

A

3

89
Q

true/false we can assume that adjacent genes are in the same reading frame

A

false

90
Q

true/false genes can be coded on either strand of the dna

A

true

91
Q
A

Molecular 3rd pt. 2 (5 decks)

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