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BIOL > Genome Sequencing > Flashcards

Genome Sequencing Flashcards

1
Q

What is the difference between a dNTP and ddNTP?

A

A ddNTP lacks a hydroxyl on the 3’ end

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2
Q

What is electrophoresis?

A

When the ddNTPs are added and bind to where it complements on the template strand then based on the different lengthed fragments the sequence can be determined

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3
Q

What do you add to a test tube for Sanger sequencing?

A

dNTP, ddNTP, taq polymerase, primers, and template DNA

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4
Q

What is the difference between the 96-well capillary and regular Sanger sequencing?

A

Sanger does not have as many wells as the capillary sequencing

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5
Q

What is an electropherogram?

A

The bar length tells you if the match is random or accurate

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6
Q

What if an electropherogram bar is short?

A

Could be random

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7
Q

What if an electropherogram bar is long?

A

The match is accurate

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8
Q

What is the shotgun approach?

A

Sequencing the genome in parts rather than as a whole

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9
Q

How is DNA fragmented?

A

Through random shearing and sonication

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10
Q

What is hierarchical shotgun sequencing?

A

The DNA is fragmented, then cloned using vectors, and then the cloned fragments are organized in a physical map to tell you the genome order

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11
Q

Why is the DNA cloned into vectors?

A

To ensure that there are multiple copies of the DNA fragment for cross comparison this can make sure that you are accurate in what you are doing

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12
Q

What is the promoter for?

A

Constituitive and inducible

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13
Q

What is the multicloning site?

A

A unique restriction site where the gene is inserted

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14
Q

What is the epitope tag?

A

Ensures that the protein is purified and localized to the right spot

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15
Q

What is the origin of replication?

A

Determines the copy number

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16
Q

What is a selectable marker?

A

Antibiotic resistance so to select for the cells you want you would add the antibiotic on the plate to kill the rest

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17
Q

What is a BAC?

A

Bacterial artificial chromosome

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18
Q

How can you use hybridization of probes to sequence a genome?

A

You take colonies from the plate using a robotic arm and a random sequence of a ssDNA probe is made and would hybridize to the overlapping fragment.

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19
Q

What is chromosome walking?

A

Where you generate a primer and the probe binds and you can determine the sequence and then you would repeat this process until you readch the end of the fragmentsa

20
Q

What is the biggest disadvantage of chromosome walking?

A

It takes too long

21
Q

How can you use restriction fingerprinting on BAC clones?

A

When a restriction enzyme recognizes its corresponding binding region it makes a double stranded break and leaves overhangs based on the overhangs of the DNA fragment you can organize the BAC clone fragments and order them

22
Q

What is whole genome sequencing?

A

You fragment the entire genome then clone it and assemble the genome without generating a physical map

23
Q

What are paired reads?

A

When an insert is sequenced from both ends using a universal primer

24
Q

What is a universal primer?

A

A forward and reverse primer with the same sequence

25
Q

What is coverage?

A

The number of times where you have to sequence a genome to have a high degree of accuracy

26
Q

What is the genome coverage assumption?

A

That the sequencing reads are randomly distributed in the genome and the ability to sequence a region of the genome does not differ

27
Q

How do you indentify overlaps?

A

They need to have >24 or more bp in common

28
Q

What is a read?

A

The product of a sequencing reaction using DNA

29
Q

What is a contig?

A

A continuous sequence made of overlapping reads with no gaps

30
Q

What is a scaffold?

A

An ordered set of contigs usually derived from mate pairs

31
Q

What is the problem with repeat regions in a genome?

A

It leads to misassembly because the system usually fails to recognize that it is in two regions

32
Q

What is an intrinsic approach to finding unknown genes?

A

Looking for the coding sequences

33
Q

What is the extrinsic approach to finding unknown genes?

A

Comparing to other genomes and looking for complementary

34
Q

What is the difference between capillary and SGS?

A

SGS
1. Don’t need vector cloning
2. Read lengths shorter than Sanger
3. Need less genomic template

35
Q

What is PCR?

A

A DNA amplification technique
1. Denature at 94 degees
2. Anneal at 54 degrees
3. Extend at 72 degrees

36
Q

What principle does Roche 454 use?

A

Luciferase assay

37
Q

Where is the DNA nicked in Roche?

A

At the beads 1 strand is stuck to the bead and binds to the linker while the other is not unless the other strand is bound to a linker

38
Q

What happens when a dNTP matches the template in Roche 454?

A

There is light from the luciferase the amount of light corresponds to how many bind

39
Q

What amplification technique is used in illumina sequencing?

A

The bridging reaction where the adaptor at one end joins to the flow cell where an existing adaptor is and generates replicates of the DNA attached to the adaptor

40
Q

How does sequencing occur in SOLiD?

A

Using an 8-mer and the first 2 nucleotides have to bind and then another 8-mer is generated

41
Q

Why is the primer in SOLiD offset by 1 nucleotide?

A

This makes it more accurate and allows for more coverage because you go over the same area more than once

42
Q

What is the difference between SGS and TGS?

A
  1. No PCR
  2. Read lengths are longer
  3. The error rate is higher and the cost
43
Q

In SMRT how is the incorporation of dNTPs measured?

A

By intensity of colour fluorescence

44
Q

What happens when the dNTPs are passed through the Oxford nanopore?

A

It generates a change in current which is then measured

45
Q
A

BIOL (3 decks)

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