Week 15 Flashcards
(96 cards)
1
Q
A nucleotide that lacks a hydroxyl group at both the 2’ and at the 3’ carbon is known as a ______.
A
dideoxynucleotide
2
Q
In massively parallel next generation sequencing technologies, how are sequencing reactions read?
A
Directly as they are run by the sequencing equipment
3
Q
In the shotgun assembly method for sequencing a genome ______.
A
genome fragments are sequenced and assembled at once
4
Q
Clone-contig sequencing requires the construction of a _______ map before sequencing.
A
Blank 1: physical
5
Q
In _______ genomics, scientists use information from one genome to infer information about a second genome.
A
comparitive
6
Q
A dideoxynucleotide ______.
A
lacks a hydroxyl group at both the 2’ and 3’ carbon
7
Q
How many human genes have no counterpart in the mouse genome?
A
300
8
Q
Choose all features common to most next generation sequencing technologies.
A
Sequencing reactions are directly read instead of using electrophoresis.
Conventional cloning is not required prior to sequencing.
Millions of sequencing reactions are performed simultaneously.
9
Q
Choose the two main approaches used in sequencing an entire genome.
A
Clone-contig sequencing
Shotgun sequencing
10
Q
According to the theory of blending inheritance, offspring were expected to be phenotypically _______
relative to their parents, which would dilute any new genetic variants.
A
Blank 1: intermediate
11
Q
Place the steps in the clone-contig sequencing and assembly method in the correct order. Start at the top.
A
- extract genomic DNA
- fragment the genome in large pieces (1-1.5Mb)
- Sequence individual 1-1.5Mb fragments using shotgun approach.
- Using genetic markers, map and construct large contigs from DNA fragments
12
Q
Which of the following are applications of comparative genomics?
A
Predict the function of a gene in an organism based on the function of a similar gene in another organism
Determine how different organisms perform similar biological functions
Determine the relatedness of organisms
13
Q
Hardy-Weinberg equilibrium requires that the population size is _____and that mating is _____
A
Blank 1: large, big, very large, very big, infinite, or infinitely large
Blank 2: random
14
Q
What percent of genes in Drosophila have counterparts in the human genome?
A
More than 50%
15
Q
The phenotype frequency is calculated by dividing the total number of individuals with a particular phenotype by which of the following?
A
The total number of individuals in that population
16
Q
If a population is in Hardy-Weinberg equilibrium for a gene with two alleles, the allele ______ of two alleles can be calculated from the phenotypic frequencies.
A
frequency
17
Q
Many 19th century scientists believed that natural selection always favored an optimal form, and therefore would tend to reduce or eliminate which of the following?
A
Genetic variation
18
Q
Consider a population and a gene with two alleles, one found at a frequency of p, and the other at a frequency of q, what equation represents the sum of all allele frequencies in the population?
A
p + q = 1
19
Q
When a population is in Hardy-Weinberg equilibrium, ______.
A
no natural selection is occurring
20
Q
If the frequency of allele A is 0.6 and the frequency of allele a is 0.4, what is the probability that an individual will receive two a alleles?
A
0.16
21
Q
Consider a population with 100 cats. If 72 cats are black and 28 are white, what is the phenotypic frequency of black cats?
A
0.72
22
Q
For a gene with two alleles, A and a, with allele frequencies given by p and q respectively, identify the chance that an offspring will inherit the following combinations of alleles in a population that is in Hardy-Weinberg equilibrium.
AA
Aa
aa
A
AA- p2
Aa-2pq
aa-q2
23
Q
The number of copies of an allele in a population divided by the total number of all alleles for that gene in a population is the ______.
A
allele frequency
24
Q
For a gene with two alleles whose frequencies are p and q, the Hardy-Weinberg equation states that the genotypes that are possible for the population are p2 + 2pq + q2, which is equal to ______.
A
(p + q)2
25
Consider a gene that has 4 alleles in a particular population: F, M, S, and VS. What is the sum of all of the frequencies of all the alleles for this gene in that population?
Blank 1: 1, 1.0, or 100%
26
Consider a gene in a population of fruit bats. There are two alleles for this gene, A and a, that occur in a population with the frequencies 0.8 and 0.2, respectively. If the population is in Hardy-Weinberg equilibrium, what genotype frequencies will be observed?
0.64 AA, 0.32 Aa, and 0.04 aa
27
The frequency of a gamete carrying a particular allele in a population is equal to which of the following?
The frequency of the allele in the population
28
For a gene with two alleles, B and b, whose frequencies are p and q, respectively, the probability that an individual receives a B allele from its mother and a B allele from its father is which of the following?
p2
29
Regarding a particular trait in a population of painted turtles (Chrysemys picta), 14% of the turtles have the homozygous dominant genotype, 70% are heterozygous, and 16% are homozygous recessive. What is the value of q2?
0.16
30
Which is a correct version of the Hardy-Weinberg equation for genotype frequencies in a population in equilibrium?
p2 + 2pq + q2 = 1
31
Select the observations that would indicate that a population is deviating from Hardy-Weinberg equilibrium.
Genotype frequencies do not match those predicated by the Hardy-Weinberg equation based on existing allele frequencies.
Allele frequencies are changing between generations.
32
You are examining allele frequencies of a gene in a population of frogs. This gene has two alleles, T and t. If the frequency of the dominant allele (T) is 0.25, then the frequency of the recessive allele (t) is
0.75
33
If the frequency of allele b in a population is equal to 0.7 then the frequency with which a gamete will carry allele b, is equal to ______
if the population is in Hardy-Weinberg equilibrium.
0.7
34
If we find evidence that a population is experiencing changes in allele frequencies from one generation to the next, we say that this population is which of the following?
Not in Hardy-Weinberg equilibrium
35
Genome-
All the genetic material of an organism- all the DNA you can find in a single cell of one animal. In the case of eukaryotes, it includes all the nuclear DNA (both coding and noncoding) and the genetic material of plastids (mitochondria, chloroplasts, etc.)- in the case of prokaryotes.
36
Genome size- Humans
```
Homo sapiens
Haploid complement (amount of DNA you find in a haploid cell): 3,200,000 bp (or 3,2 Gb )
23k genes (allegedly…)
```
Mammals
If we compare the genome size in mammals, humans are in the middle of the range of sizes. For example, the smallest genome in mammals is found in a group of bats. The largest mammal genome belongs to rats.
37
Genome size: largest animal
Protopterus aethiopicus
AKA marbled lungfish: 133 Gb
x400 compared to fugu, x40 compared to human
38
Genome size: smallest animal
```
Pratylenchus coffeae Nematode
19 Mb (x170 smaller than human genome)
```
39
Largest genomes ever
```
Paris japonica, octoploid and suspected allopolyploid hybrid of four species
150 Gb (x50 compared to human)
```
40
Smallest genomes
```
Bacteria
Carsonella ruddii
160 Kb
182 genes
Endosymbiont of sap-feeding insects
```
41
What is the C-Value paradox?
•Genome size does NOT increase with perceived complexity of organisms.
In the graph you can see a list of organisms from bacteria to fungi etc and you can see the range of genome size, as you can see you can find much larger genomes compared to mammals for example you find groups that have larger genomes, like in the case of flowering plants etc which are larger than the mammal ones. So there was this paradox that those genes, those genomes were larger than expected. This is called the C-value paradox, the c-value is this measure of the genome size that was based basically on staining the genome, the DNA in the cells of an organism and quantifying the amount of DNA. So people were using this approach and they were seeing that the C-Value, the amount of DNA they were finding in an organism was not matching their perceived complexity.
42
Number of genes is _____correlated to ______ size
not
| genome
43
How does the Eukaryote genome produce more than one protein per gene?
•C. elegans has 100 Mb and 20k genes
Drosophila has 165 Mb and 14k genes
•Eukaryote genomes can produce more than one protein per gene because of alternative splicing of RNA transcripts
So what's happening is that more complex organisms really have more genes, what's happening is that there is no correlation between the number, the amount of DNA and the number of genes in a genome. This is partially true, it's true that there is no correlation between genome size and the amount of genes, the gene complement that you find in organisms, however, it's not true that more complex organisms have more genes.
44
“Paradox” also applies to
number of genes
45
What are aspects of Bread wheat?
```
Hexaploid, cross between tetraploid wheat and diploid grass
100k genes (28k, 38k, and 36k respectively)
```
46
Parasites usually have
small genomes
47
Why do parasites have small genomes?
•Smallest gene complement in animals: mesozoans, 9k genes
•Parasites have lost many structures, including the digestive system
Its the only group of animals that are intracellular parasites of other animals. Its able to enter the cell of another animal and become a parasite.
48
Outline some aspects of genome Architecture
Archaea are more closely related to eukaryotes
Prokaryotes contain two lineages- bacteria and archaea
Prokaryotes have a smaller genome usually than eukaryotes.
The gene density- higher in prokaryotes meaning that the genes are more close to each other.
49
Coding vs noncoding DNA
* Mammals have the lowest gene density, or number of genes in a given length of DNA- so that means there is plenty of DNA that is not coding for a protein.
* Sequencing of the human genome reveals that 98.5% does not code for genes (genes meaning proteins, rRNAs, or tRNAs). Most of the genome is full of things that do not have instructions to code for a protein. There might be regulatory regions but there are actually other things.
* 25% of the human genome codes for introns and gene-related regulatory sequences (e.g. enhancers, promoters,…).
* What about the rest…?
50
Genome elements in eukaryotes -
Protein-coding genes
Non-protein-coding DNA
Repetitive sequences
51
What are Protein -coding genes- Exons, regulatory sequences and introns?
Protein-coding genes: not only exons and introns, but also enhancers, promoters, the 5' upstream untranslated region (5' UTR), 3' region downstream from the stop codon (3' UTR), the poly-A tail.
You have enhancers/silencers which are regions of the gene that can boost or repress the amount of gene that is being expressed.
The promoter that can regulate transcriptional state of protein coding genes, on and off.
The UTR, the exons in the red and the introns in grey, stop codon, the three prime UTR, silencer and you have all these processes that we have see. - all part of protein coding gene.
52
What is Non-protein-coding DNA?
Noncoding DNA
Genes- sections of the DNA that are going to be transcribed to RNA and perform a function. But the function is performed by the RNA itself. Those genes are not translated, the RNA of those genes is not translated to protein.
Noncoding DNA: non-repetitive DNA that mostly codes for RNA genes (non-coding RNA or ncRNA), RNAs with a function.
Small ncRNA:
•rRNA: Ribosomal RNA
•tRNA: Transfer RNA, brings amino acids to the ribosome.
•snRNA: Small nuclear RNA, in spliceosomes to process pre-mRNA.
•snoRNA: Small nucleolar RNA, process and construct the ribosome.
•gRNA: Guide RNA are used in RNA editing.
•miRNA: Micro RNA, ~24 nucleotides RNAs used in gene silencing.
Long ncRNA: over 200 nucleotides, regulate gene expression
53
There are many more ______ ______ genes in the genome than protein coding genes. In the case of humans we have around ______ protein coding genes. However, if you look at the non-coding elements of the genome there are around ______ non-coding genes. You can split those with long noncoding RNAs.
non-protein coding
33000
36,000
54
What are repetitive sequences?
Are a big component of eukaryotic genomes, this is actually one of the main differences with prokaryotes that prokaryotes are very economic genomes in the sense that they do not have stuff in the, while our genomes do.
I.Unrelated to Transposable Elements
II.Related to Transposable Elements
I.Unrelated to Transposable Elements:
•Sequences repeated next to each other (tandem repeats)-
•Originated by strand slippage during DNA replication
•Minisatellites (repeats of 10 -60 bp)- small repeats
•Microsatellite (less than 10 bp)- longer repeats
55
What is DNA strand slippage?
You have normal replication- along here you have the template strand that just by nature is very repetitive. Then if everything was going well, then the new strand will have a complementary sequence to this CTG.
But sometimes we have a mistake and as all those units are complementary, you might have that in one round of replication the new strand will make a loop, because this repeat here the GAC is going to slip and pair with the repeat here, excluding this repeat in the middle. What will happen now is the replication round is finished you will have that one of the chains will have an insertion because now you have all the same repeats you had before and an extra 1 because this one was left out of the loop so we’ll have an additional repetition, while the other outcome of this same slippage event will be a normal length, but the slippage can also happen in the template. In that case what is happening is that this repeat here is pairing with a repeat everywhere and shouldn’t have been.. So the CTG should have been paired with this GAC here and what's happening as a consequence is that you have one of the units that is left out in a loop and now in this case what you have is that at the end you will have at one of the outcomes or one of the chains is going to be a deletion because you will lose this, one of the repeats in the chain, while in the other outcome you will have a normal length. This is when you have a diversity in the repeats.
56
What are microsatellites?
* Microsatellites are often referred to as short tandem repeats (STRs) by forensic geneticists, or as simple sequence repeats (SSRs) by plant geneticists
* 1000 Genomes Project found 700,000 STR loci across more than 1000 individuals
* Each locus shows high diversity in the number of repeats in human populations
Microsatellites are used for DNA profiling/fingerprinting
* PCR is done for 13-20 different STR loci at the same time
* Run in an agarose gel, STR locus with more repeats will be heavier.
57
What are repeated sequences?
3.Repeated sequences:
II.Related to Transposable Elements: repeated sequences spread across the genome (interspersed repeats).
Viral sequences but also transposons (transposable elements or TEs)- are the origins of those repeats, so viruses enter the cells of the host and then integrate their sequence in the genome and then if they do this multiple times you end up with repeats in the genome and those viruses can integrate in different parts of the genome, so you will have those interspersed repeats, but they can also be caused by those enigmatic rogue elements of the genome called transposons.
58
What are transposons?
are genomic accidents, they are little bits of the genome that has the ability to auto-replicate so by accident they have acquired this ability to make copies of themselves and insert those copies in other parts of the genome.
59
What are the two types of transposons?
There are two types:
•DNA transposons, Cut & Paste jumps
•RNA transposons, Copy & Paste jumps
•Transposons, or transposable elements, or mobile elements, or jumping genes.
60
What did Barbara McClintock discover?
Barbara McClintock discovered them doing breeding experiments with Indian corn. Nobel in 1983.
McClintock identified changes in the colour of corn kernels that made sense only if some genetic elements move from other genome locations into the genes for kernel colour
These transposable elements move from one site to another in a cell’s DNA; they are present in both prokaryotes and eukaryotes
61
Movement of Transposons and Retrotransposons-
DNA Transposons move by means of a DNA intermediate and require a transposase enzyme. They move by Cut & Paste.- they are a sequence of DNA and they require a transposase enzyme, a transposase enzyme is an enzyme that will bind to the DNA, will recognise the transposon sequence, will bind to a DNA and it will cut the sequence out of the genome. Then now this DNA sequence accidentally will be able to insert itself in another part of the genome. Which can be messy because this may leave some DNA may be broken behind but usually will get repaired by the DNA machinery but not always.
---RNA transposons, or Retrotransposons, move by means of an RNA intermediate, using a reverse transcriptase. They move by Copy & Paste. Then we have RNA transposons- you have a sequence in the DNA that is going to be transcribed to mRNA and now the transposase will take this RNA sequence and will insert it in a new region of the genome. But the original sequence stays where it is.
62
DNA transposons
TIR:
TSD:
TIR: tandem inverted repeats
TSD: tandem site duplications
63
How do these DNA transposons work?
Mechanism:
1. Two transposases (in green) bind to TIR.
2. DNA cleavage.
3. The DNA-transposase complex inserts its DNA cargo at specific DNA motifs elsewhere in the genome, creating new short TSDs upon integration
64
What does the sequence of transposons do?
The sequence of the transposon that includes the transposase. The Transposase enzyme is flanked by tandem inverted repeats, those are sequences of repeat letters that are inverted on one side compared to the sequence on the other flanking side and then you also find those tandem site duplications next to the tandem inverted repeats. So now this transposase will be transcribed and it will be translated and then the protein will be floating in the nucleus and it will bump into the DNA until it recognises two transposases, and will recognize those tandem inverted repeat sequences. Now it will bring together the two sequences of DNA and it will cut the transposon sequence that's in the middle, and now this sequence will go away and you will have DNA ligase coming that will ligate them all again. You will be left with the two TSDs you had here- generating a repeat.
65
What are Retrotransposons?
You have the transposon sequence here that will be translated to mRNA but now this mRNA wont produce a protein that will be bumping into DNA like it was happening with the DNA transposons. Now this RNA molecule will be retrotranscribed by reverse transcriptase and this is again an enzyme that may be found in the retrotransposon but is typically found in RNA viruses that they need. Will be retrotrancribed by reverse transcriptase that will generate a DNA strand that will be a single one that we can transcribe to a double copy of a ds DNA. This copy of the transposon is able to insert in another part of the genome and now you have these two copies of the transposon.
66
Two methods of transpositions?
1) Cut and paste mechanism- DNA Transposons and Cleavage of original copy.
2) Copy and paste- RNA transposons and DNA > mRNA > cDNA
67
What are Alu Elements?
* In primates, a large portion of retrotransposons consists of a family called Alu elements
* Alu elements are the most abundant gene in the human genome, 1,000,000 copies per genome, 10% of the genome
* Many Alu elements are transcribed; some are thought to help regulate gene expression (rich in CpG, important in epigenetics)
68
Transposable Elements and Genome Evolution-
•Multiple copies of similar transposable elements may facilitate recombination between different chromosomes- the long stretch of DNA that was highly repetitive can be found in two different chromosomes or two different parts of a chromosome. So that means within meiosis and mitosis, now those non-homologous chromosomes will be able to recombine.
69
Provide an example of how these uneven crossovers might occur?
An example of how these uneven crossovers might occur- so here you have two pairs of homologous chromosomes that are pairing in meiosis and one is from the dad one from the mum. If you look into the sequences here you have this is 1 chromatid from one of the chromosomes and here you have a gene and in yellow you have this transposable element for which there are two copies and then you look at the mum chromatid and you find again the same transposable element in the same positions. Where you have recombination what might happen is that by accident this region of the, one of the parental chromosomes is going to pair and recombine with this region in the chromatids of the parental which is wrong. You are going to end with one chromatid that has an extra bit of DNA and the other one that is missing a bit.
70
* Insertion within a protein-coding sequence may ____ protein production.
* Insertion within a regulatory sequence may _____ or ______ protein production
* Transposable elements may ____ a gene or groups of genes to a ____ position.
* Transposable elements may be the cause of many gene ______mechanisms: DNA methylation, ncRNAs, etc.
* In all cases, changes are usually detrimental but may on occasion prove ________ to an organism
```
block
increase or decrease
carry
new
silencing
advantageous
```
71
Junk DNA or selfish DNA-
* Noncoding DNA that (allegedly) has no function
| * 68% of genome is repetitive sequences scattered randomly
72
Large genomes are usually full of
repetitive elements
73
•Ambystoma mexicanum, AKA axolotl-
* Salamander (amphibian)
* 32 Gb (Feb 2018)
* 66% genome is repetitive
* Huge introns, x25 larger than human introns
74
•Schmidtea mediterranea
| AKA planarian
* Flatworm
* 800 Mb (Feb 2018)
* 62% genome is repetitive
* Huge retrotransposons insertions, over 30 kb long (5-10 kb in vertebrates)
75
Large genomes are usually are also the result of
polyploidy
76
What is epigenetics and what mechanisms are included?
Epi- means “on top”
Heritable changes of genetic information not caused by changes in the DNA sequence. Mutations change the DNA sequence, epigenetics does not.
```
Mechanisms include:
Epigenetic marks (histone modifications and DNA methylation)
RNA interference (ncRNAs)
```
77
Originally epigenetics in the first incarnation it was coined to talk about this ___ _____ that explains during cell ______ during _______ development.
cell memory
differentiation
embryonic
78
We start off being a _____cell eukaryote then the single cell starts to divide more and more until you end with _______ of cells. So all those cells have the _____ genome and they have the same genotype. However, each expresses different _______ and this is because there is a differential gene _______.
```
single
thousands
same
phenotypes
regulation.
```
79
Some genes are switched on and off in different cells. _______ factors do not explain how one cell remembers when it is divided into 2 daughter cells, or how those two daughter cells remember the transcriptional state from the previous generation of cells.
TRANSCRIPTION
80
Differential gene expression: Phenotypic plasticity
They all have the same genome but they have very different phenotypes and this is because the epigenetic mechanisms that are going to activate and inactivate different genes in the different members of the hive. Phenotypic plasticity- meaning that for one single given genome you have a different phenotype that can be expressed thanks to epigenetics.
81
Why is there differential gene expression?
Different epigenetic mechanisms.
In mammals all those epigenetic mechanisms, chemical modifications of the DNA or histones. All those epigenetic mechanisms, modifications they get reset when you make your gametes. So that means that there is no way in mammals to pass those epigenetic changes from one generation to the next one. In invertebrates- like nematodes there are no reset mechanisms during one generation to the next and we have evidence to show that epigenetics changes can be inherited in up to 13 generations.
82
What is histone modification?
Chemical modifications of histones change the folding of DNA
Histones are proteins that together with the DNA they form the chromatin. So you have the chromosome you zoom in, you have the chromatin that are made by those nucleosomes that are those histones proteins that are interlaced with DNA. A gene that is found in a chromosome region in order to be transcribes it needs to be in a chromosome region that is not compacted, that needs to be chromatin that is very relaxed, so it is physically available so the transcription factor can work on that gene. Thats in contrast with chromatin that is highly compacted, in which the DNA is so compact that all those proteins cannot go in and start to transcribe genes so that means that different cell types are going to have different chromatin states. Means that in some cell types you will have some buts o chromatin that are relaxed and those are the bits that contain the genes needed for that cell type. But in other cell types the same genes will be closed and other ones will be open and more relaxed and more available.
83
What is one way to control chromatic compaction?
One way to control chromatin compaction is through the histones, so we can do chemical modifications of histones, they have tiny tails at one of the ends, the terminal end of the histones that protrude from the nucleosome. There are proteins that are able to interact with this histone tail and change the chemical composition, like doing acetylation for example. So when the histone tails are modified this is going to change the physical chemical properties for the histone its going to change the structure and this is going to make the chromatin become relaxed or compact.
84
What are two examples of DNA modifications?
Two examples- you have one case in which the histone tails have a methyl group that makes them more compact and now we want to transcribe a gene that is in this bit of DNA, we can not transcribe it, we cannot activate this gene its not accessible, its physically blocked by the sounding chromatin and transcription factors cannot access it. Bottom shows the opposite case- we have that the histone tails are modifies with an acetyl group and now we’re going to be able to make the chromatin relax. So we now have a gene here that is going to become accessible and now transcription factors and all the machinery involved in transcription, will be able to access this gene and activate it. And its not only one gene its all the genes in that area would become available, which means that many times you have that in the genome, genes that are involved in similar biological functions or similar cell types are found together.
85
What is DNA methylation?
Addition of a methyl group (CH3) to DNA makes it inaccessible.
Have specific proteins/enzymes that go to those DNA marks.
Regions rich in CGs (CpG islands) are NOT methylated
CpG is shorthand for 5'—C—phosphate—G—3'
They act as a repression system- repress genes. They are basically physically blocking the access of transcription factors and the transcription machinery to the DNA. they are blocking transcription. So when the genome has a region that is methylated, the genes that are in that region won't be expressed. The genome in this case of mammals at least, when it wants to protect an area, a region of the genome against methylation, so when it wants to keep a region of the DNA active in a transcriptional sense, what you find are those CPG islands. They are the regions rich in cytosines and guanines- those regions are never methylated, those enzymes that methylate DNA never act on those CPG regions. That means that this region will be accessible- found in the promoter regions of genes.
86
Histone modification is going to regulate ______compaction. If chromatin is compacted, genes are ___ expressed. If chromatin is relaxed, then genes can be _______.
chromatin
not
expressed
87
What are aspects different levels of gene regulation?
Dynamic of DNA methylation during mouse embryonic development.
The graph tells us the % of methylation- differences in methylation and this is because this level of gene regulation is very dynamic in the same sense that you might have transcription factors going up and down during development, it can happen the same with those epigenetic marks.
88
What is genomic imprinting?
* Gene expression of an allele depends on parent-of-origin
* There are about 150 imprinted genes in mouse and about half that in humans
* Example: insulin-like growth factor 2 (IGF2) only allele inherited from the father is expressed
89
Where can X inactivation be observed?
Calico cat
This happens in females when you have two X chromosomes and now in different cell types and different cells, there's going to be a random inactivation of one of the X chromosomes that is going to become methylated and it's not going to express the genes in there. So that explains these patterns of different fur colours because some of the cells, for example in this case that are orange, have suffered the same random inactivation of the X chromosome while the others have been activated on the other one.
90
What is the first generation of sequencing during genome sequencing?
•Sanger sequencing- a modification of the PCR by using modified DNTPs that made inflorescence that terminates the extension of the template chain. So we do a PCR with a mix of those special DNTP’s the terminators and normal DNTPs and then that will generate sequences of different lengths that will be the last nucleotide on each of those sequences will be one of those inflorescences.
•Optimised to have a priori genetic information (primers for PCR), not high throughput
•The human genome was sequenced this way ($1 billion, 15 years, 18 countries)
First-generation DNA sequencing technologies. Example DNA to be sequenced (a) is illustrated undergoing Sanger (b) sequencing. (b): Sanger's ‘chain-termination’ sequencing. Radio- or fluorescently-labelled ddNTP nucleotides of a given type - which once incorporated, prevent further extension - are included in DNA polymerisation reactions at low concentrations (primed off a 5′ sequence, not shown). Therefore in each of the four reactions, sequence fragments are generated with 3′ truncations as a ddNTP is randomly incorporated at a particular instance of that base (underlined 3′ terminal characters). (d): Fragments generated from either methodology can then be visualised via electrophoresis on a high-resolution polyacrylamide gel: sequences are then inferred by reading ‘up’ the gel, as the shorter DNA fragments migrate fastest. In Sanger sequencing (left) the sequence is inferred by finding the lane in which the band is present for a given site, as the 3′ terminating labelled ddNTP corresponds to the base at that position.
91
What is shotgun sequencing?
1) cut the DNA into overlapping fragments short enough for sequencing.
2) Clone the fragments in plasmid or other vectors.
3) Sequence each fragment
4) Order the sequences into one overall sequence with computer software.
92
Describe shotgun sequencing in more detail.
The way they did the human genome with Sanger sequencing, because of course without, we don't know the sequence of the genome. How do we sequence those bits for bits we don’t know the sequence and therefore we cannot design a primer. They did something called shotgun sequencing, so here we have the DNA in that case one chromosome, and now you are going to breakdown this chromosome with enzymes and they do this randomly, the nucleases that are used in the shotgun sequencing, they cut the sequences randomly, meaning that for example you are going to have different bits of the same region, that one bit will be found in one fragment, another will be found in another fragment. It's called shotgun because it's like taking a shotgun and shooting the chromosome to break it down. We can take all of those pieces and we can clone it in a vector, we can put it on a plasmid, we do know the sequence of the plasmid. Now we have those fragments of the human genome of unknown sequence, but they are integrated in a plasmid with a known sequence. Now we can design primers based on the plasmid sequence to sequence this bit of the DNA. as you are digesting, you are cutting down the DNA randomly. Then you need an additional step of putting back everything together. So you want to reconstruct the sequence, so you are going to look at which bits overlap with each other and you will try to reconstruct how it looks like.
93
What is second generation sequencing?
It's already designed to sequence genomes in the sense that it's going to take advantage of this shotgun strategy, and it's going to take it to a new level. And it's going to increase the output of sequences dramatically.
* Example: Illumina- used for RNA sequencing and quantitative sequencing. You can extract all the messenger RNAs of tissue one, two etc sequencing them independently and you are able to see the copies
* Do NOT need a priori genetic information- it's going to sequence random bits of DNA using random primers, so you’re using primers generated randomly, you don't need to know which sequences are present in the genome because it's going to generate millions of different primers that you can use to perform the sequencing.
* High sensitivity, quantitative method
* A human genome can be sequenced by $1,000, under a week, in a single lab
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What is third generation sequencing?
The focus is on having lone molecules because of the problems we were mentioning with transposable elements, repetitive sequences that are a problem in the genome.
•Example: Nanopore
•Do NOT need a priori genetic information
•Focus on single molecule sequencing
•No need for shotgun sequencing
The whole technology is based on a protein from bacteria, so its a pore protein that bacteria usually have in the membranes of their cells and this pore protein that gives the name Nanopore is embedded in a circuit. It will take one molecule of DNA and make it go through the pore. Now as the nucleotides pass through the pore, they’re going to change the confirmation of the physical/ chemical properties of the protein of the pore. Will change the electrical charge of this pore.
* Our ability to explore all areas of nature has improved incredibly
* This change has been caused mostly by Next Generation Sequencing
* Impact not only on molecular medicine, but also in ecology, zoology, botanics, microbiology, and evolution
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What is genome sequencing?
both second and third generation sequencing technologies are sequence blind, meaning that you don't need to have any knowledge of what you’re going to sequence because you don't depend on a PCR because you have these random amplification approaches.
Microbiome- came from this technology.
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Phylogenomics: more genes/ more taxa
When we are trying to reconstruct the evolutionary history of organisms using genome level information , it has dramatically changed our understanding of evolution.
