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Genetik > 2 > Flashcards

2 Flashcards

1
Q

what is an outgroup?

A
  • a group, closely related to the group being studied
  • but: not part of the group
  • used as a reference point for making inference on evolutionary relationships within ingroups
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2
Q

closed system

A

assumption that no demographic changes occur (birth, death…) during period of study

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3
Q

open system

A

assumption that demographic changes occur during period of study

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4
Q

different kinds of samples we can take from marine organisms

A
  • Fin clips
  • Muscle
  • Parts of the body
  • Blood
  • Otoliths
  • Scales
  • Water
  • Gut and stomach contents.
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5
Q

characteristics of an ideal marker

A
  • Random distribution in the genome (whole genome cover)
  • Highly polymorphic (so the same marker should have multiple alleles and so multiple forms)
  • Codominant, so we can distinguish between homo (only 1 variant of the marker) and heterozygotes
    (2 variants of the marker) individuals
  • Inheritable (vererbbar)
  • Neutral (so not under evolutionary forces pressure) and sex/age independent
  • Stable (easy replication) and easy to monitor
  • Frequent and present in all tissues
  • Universal application, so all the scientists in the world could use it
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6
Q

what is DNA sequencing?

A

process of determining the sequential order of the 4 nucleotides

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7
Q

methods of DNA sequencing

A
  • first generation sequencing
  • PCR
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8
Q

what does PCR do?

A
  • makes many copies of a part of a genome
  • allows to analyze different kinds of molecular markers
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9
Q

PCR-technique

A

3 main phases

(1) denaturation: opens double-helix (95°C)
(2) annealing: different forward- and reverse-primers are created and inserted (68°C)
(3) extension: single dNTP’s + polymerase added to solution –> enzyme binds to primer and starts elongation (72°C)

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10
Q

Sanger Sequencing

A

3 main phases of PCR

(1) denaturation: opens double-helix (95°C)
(2) annealing: different forward- and reverse-primers are created and inserted (68°C)
(3) extension: single dNTP’s + polymerase added to solution –> enzyme binds to primer and starts elongation (72°C)

Sanger did more:
- he inserted dNTP’s and ddNTP’s ( = di-deoxy nucleotide triphosphate) with fluorophore (each base with specific color)
- they are modified nitrogen bases without oxy-group on 3’
- it causes the detachment of the enzyme polymerase when it incorporates this base in the filament

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11
Q

after sanger sequence, what do we get as a result?

A
  • thousands of fragments
  • all different lengths
  • all with a fluorophosphated ddNTP at one end
  • fragments will be denatured again
  • then we get single mononucleotides with fluorophore
  • the fragments of different lengths are put in a gel
  • they travel towards +Pole
  • the shortest fragment travel the fastest and longest
  • a computer can catch the fluorophore-light linked to the fragment and reports the emitted wavelength
  • result: chromatogram
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12
Q

pros of the Sanger Sequencing Method

A
  • fast
  • automatic
  • relatively cheap
  • negative: can produce errors
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13
Q

example of a good chromatogram after Sanger sequencing

A
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14
Q

example of a errors in chromatogram after Sanger sequencing

A
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15
Q

SangerSequencing (early method)
pattern on electrophoretic gel

A
  • 4 columns (G C A T)
  • slide must be read from bottom to the top
  • bottom: fastest and shortest fragment
  • top: longer and slower moving fragments
  • we can reconstruct the sequence of nucleotide that composes DNA
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16
Q

what is “sequencing by synthesis”?

A
  • a next generation sequencing method
  • improved Sanger sequencing method
  • can perform millions of sequence reactions at the same time
17
Q

second generation sequencing

A
  • illumina NGS
  • able to perform short to medium reads: up to 500bp
  • first step: preparation of DNA, called library

preparing a library:
1. extracting DNA from a sample
2. fragmenting it
3. repairing the ends
4. ligating adapters
5. and amplifying the fragments
6. result: a collection of DNA fragments with known sequences and uniform characteristics

18
Q

third generation sequencing

A
  • Oxford Nanopore NGS
  • able to perform long reads: more than 1 kilo bases
  • first step: preparation of DNA, called library
19
Q

what means “prepare a library”?

A
  1. fragmenting the genomic DNA
  2. ligating adapters to the fragments (for identification)
  3. and amplifying the resulting library through techniques like PCR, creating a collection of DNA fragments ready for high-throughput sequencing
20
Q

differences between Sanger and NGS

A
  • NGS is similar to Sanger because we always sequence fragments, but it allows millions of fragments to be sequenced in a single run (while Sanger produce only 1 forward and 1 reverse read)
  • Newer NGS are becoming common in labs due to their higher capabilities and lower costs per sample than Sanger method
  • Sanger is perfect for long reads, for sequencing single genes, for sequencing 1-100 amplicon, for microbial identification and for NGS confirmation
  • NGS are good for sequencing hundreds to thousands of genes or gene regions simultaneously,
  • NGS are good for sequencing samples that have low input amounts of starting material and are excellent for finding novel variants by expanding the number of targets which are sequenced in a single run
21
Q

negative aspects of second NGS?

A
  • areas that are G and C rich are regions where many errors occur
  • expensive
  • time-consuming
  • needs amplification of PCR
  • able to read produce only short reads (500bp) which has 3 implications (picture)
22
Q

third NGS pros

A
  • does not need PCR amplification: no PCR-errors associated
  • can sequence single and very long DNA fragments
  • produces therefor long reads
  • ONT (oxford nano technology) can directly read the sequence of a single strand DNA molecule
  • can read filaments in real time
  • can be applied for detection of DNA and RNA and proteins
23
Q

When do I chose which method?

A
  • depending on (1) number of apples (2) number of genes (3) purpose of our study
  • if few samples are enough for our study and we have a low number of genes: Sanger
  • if number of genes increases and we want to discover a whole gene: NGS
24
Q

what is NGS nowadays extremely used in?

A
  • clinic analysis
  • food traceability
  • metagenomics (eDNA for monitoring)
  • population genomics (to discover SNP’s)
  • genome assembly
  • on site analysis (direct barcoding in field)
25
Q

table (YouTube) for Sanger and NGS

A

Genetik (11 decks)

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