Search
News

Page 1 of 2  > >>

Apr 4, 2013
Apr 4, 2013
Jan 7, 2012
Sep 2, 2011
Contact Info

Björn Usadel.
Worringer Weg 1
52062 Aachen
Germany
Email: usadel /ta/ mpimp-golm.mpg.de
usadel /ta/ bio1.rwth-aachen.de

Phone: +49 241 80 26634
You are here: Teaching » MoGe I » MogeI Quizz

Moge I Quizz

Lecture 1 Introduction

How can the genome size of an organism in MB be estimated using a simple experiment?

 

This can be achieved via the c-Value 1/45

 

 

Are there genomes larger than the human genome?

 

There are many even in relatively "simple" organisms 1/49

 

 

If you compare our ability to sequence cheaply how do you think that will affect (bio) informatics?

 

Sequencing will become (is) cheaper than storage so computer science has to be pushed and find new solutions. (Often a suggestion is throw the data away but the cost for the experiment is not taken into account)e.g. 1/22

 

 

Lecture 2 Gene Structure

Name at least four different RNA species produced in the eukaryotic cell?

 

mRNA, rRNA, tRNA, snRNA, miRNA, ... 2/4

 

 

Name at least four different RNA species produced in the eukaryotic cell?

 

mRNA, rRNA, tRNA, snRNA, miRNA, ... 2/4

 

 

How does an RNA polymerase "know" which strand to read from?

 

this depends on the promoter (more detailed answer needed!) 2/9

 

 

Describe a very basal sigma70 E.coli promoter and name at least one consensus sequence?

 

-35 and -10 element 35 and 10 bases upstream of promoter. TTGACA TATAAT Some might have an additional UP element 2/10

 

 

Explain a sequnece logo based on frequency?

 

See picture, you would rather see a figure and would have to explain it 2/15

 

 

Explain a sequnece logo based on information content?

 

See picture, you would rather see a figure and would have to explain it. It is enough to have a rough idea about pseudocounts 2/15

 

 

Which fraction of the eukaryotic genes seem to contain a TATA box?

 

only about a third, but almost all might have a TATA or TATA like box 2/21

 

 

Depict an eukaryotic promoter with three typical upstream elements without consensus sequences?

 

see figure 2/23

 

 

What does TBP (as part of TFIID) stand for?

 

TATA binding protein 2/24

 

 

Explain the sequence for gene transcription involvidn TFIID,B,H?

 

see 2/26-2/28

 

 

Explain a gel retardation assay?

 

Fragments are incubated with and without nuclear proteins that might bind. In the fraction with potential binding protein a band is “retarded” moved to apparently higher size 2/31

 

 

Explain a DNA footprinting assay?

 

DNA fragments are treated with and without DNA binding protens; afterwards the fragments are digested with DNAse, binding proteins protect : a footprint is visible on the gel 2/32

 

 

You have found a likely promoter fragment, how would you identify a binding protein?

 

Explain either 2/33 affinity purification, 2/33 clone based and/or 2/34 Y1H

 

 

How can you define the binding site of a transcription factor?

 

Explain either SELEX 2/39 aand/or PBM 2/40

 

 

How can you find (alternative) splicing events using only in-silico public data?

 

ESTs can be mapped onto the genome and splice sites be found 2/52

 

 

Describe what ESTs are?

 

See figure 2/53 for an overview

 

 

Draw a scheme of how an (eukaryotic) gene might look like and what we can identify?

 

Figure at the end of the lecture: Cis Elements, core cis Elements Introns, alternative Exons, Exons, splice modifiers e.g. ISE

 

 

Lecture 3A Measuring Transcripts

If you want to measure the amount of RNA by means of q-RT-PCR what is an important first step?

 

The RNA needs to be reverse transcribed into cDNA first, e.g. using an oligo dT primer 3A/2-3

 

 

How can classical PCR be used to get an idea about DNA amount?

 

For each sample a “constitutive” product is monitored as well. The PCR reaction is Stopped after various cycle numbers (or just at one cycle number) or competitive PCR see question 3A/6

 

 

What is the problem with the reference gene approach for determinging RNA amount?

 

The reference gene is used to standardize the amount of RNA, this would assume that the expression of the referennce gene is constant under the assayed conditions, whcih might not always be the case 3A/6-7

 

 

Describe competetive PCR?

 

A standard which is very similar to the target (e.g. some differnet length but using the same primers!) is added in known amounts to the reaction mixture, where band intensities are equal one can dtermine the amount 3A/8

 

 

Describe the Sybergreen pcrinciple

 

Sybergreen (liek Etidiumbromide) interacalates in double stranded DNA, which is produced by PCR, the more dsDNA is made, the higher the absorbance. The reaction mix is almost like normal PCR+ Sybergreen 3A/10

 

 

Describe the TaqMan pcrinciple

 

Probes which hybridize to ssDNA are added to the PCR mix, these have two dyes, one absorber and a quencher, which quenches the signal by resonance transfer if both probes are close. Once polymerase comes by the probe is depolymerized thus the probes are far from each other => signal increases 3A/9-10

 

 

What is the CT value in real time qRT-PCR?

 

The number of cycles that have been run when a a certain Fluorescence threshold has been reached 3A/11

 

 

Explain the Delta CT method for real time qRT-PCR data evaluation?

 

The Ct values of gene of interest and a "standard" are simple subtracted 3A/13

 

 

Name at least 6 golden rules for real time qRT-PCR?

 

At least three BIOLOGICAL reps, High quality RNA, Digest RNA with DNAse I to remove genomic DNA, Use a good reverse transcriptase with no RNase H activity, Test cDNA, Design good primers, Standardize and use a master mix, Test at least 4 potential reference genes, Perform real time PCR on test and reference genes at the same time (and use melting curves), One should determine the best reference genes, Calculate transcript abundance using efficiency 3A/18

 

 

Explain the workflow for a two color microarray?

 

See figure 3A/24

 

 

Explain how Affymetrix microarrays are made (specifically the photolitographic process)?

 

wafer is Silanated-> start,Linker is added, UV light deprotects linker but not where a mask is shielding UV light, nucleotide with protection is added, UV light with shileding and repeat, finally capping reagent is added 3A/28-32

 

 

What are PM and MM probes on the Affymetrix chip?

 

perfect match and mismatch probes, the PM are complemnetary the MM have a mismatch in the 13th of 25 nucleotides 3A/34,36

 

 

Why is array normalization necessary?

 

This is necessary in order to compare arrays and thus samples, as one might have different amount of RNA extracted, a different laser internsity, different washing conditions etc

 

 

Which major assumption underlies most array normalization procedures?

 

One big assumption is that not too many genes are changing, or in other words most genes stay more or less the same 3A/42

 

 

How can one use the different probes on an Affymetrix array determine RNA quality?

 

One knows where the probes are on a gene and can order them 5' to 3' Synthesis often starts from the 3' poly A tail so one can see how the signal changes from 3' to 5' 3A/54

 

 

Lecture 3B Measuring Transcripts

Which kind of arrays would you use for gene discovery?

 

One could use Tiling arrays, which "tile" the genome by setting probes very close to each other 3B/5ff

 

 

When you have one probe A having a higher intensity than another probe B, does this necessarily mean that what is measured by A is higher than that measured by B?

 

No whilst the intsensity of a probe should be somewhat proportional to the substance it measures, different probes might behave differently 3B/10

 

 

Why might you see a signal for a specific probe on an array, even though the gene it should measure is not active?

 

This could be due to cross hybridization to e.g. a gene from the same gene family. (Background noise might be another explanation) 3B/15

 

 

Name some Tiling array applications, categorized into three groups?

 

poly A hyb: Gene discovery, gene validation, differential expression, expression height; DNA hyb: copy number variation, CGH, genotyping; Chromatin IP: TF binding sites, methylation, histone modification 3B/18

 

 

What does CGH stand for and how can it be used?

 

Comparative genomic hybridization, to detect differnt copy numbers of genomic regions 3B/31

 

 

What is ChIP-Chip, explain how it is done?

 

Chromatin Immunoprecipitation with Chip hybridization see figure 3B/40

 

 

How can one use antibodies against certain histone modifications to crack the histone code?

 

By ChIP-Chip see the end of lecture 3B

 

 

Lecture 3C Measuring Transcripts

What is GO and how can it be used to understand array data?

 

This is the Gene ontology, an ontology to classify genes. One can use it to look for groups of genes which are commonly up or down regulated 3C

 

 

What is a controlled vocabulary and why is it useful?

 

A controlled vocabulary is giving only a certain set of "controlled" words that should be used to describe something. As these are controlled there is no ambiguitiy and we know what we speak of. 3C

 

 

What is an ontology and how is it useful?

 

XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXxx 3C

 

 

Be prepared to interpret a tree?

 

be mindful about the topology 3C/18

 

 

Be carefull 3C doesn't bring in a lot of things but can be used to make sense of data.

 

Be carefull 3C doesn't bring in a lot of things but can be used to make sense of data.

 

 

How can correlation be used to learn something about genes?

 

If genes are correlated accross a large number of arrays, the MIGHT have a similar function 3C/25

 

 

Lecture 4 Transposons

Be prepared to interpret Figure 4/2 showing a C0T graph?

 

Be prepared to interpret Figure 4/2 showing a C0T graph?

 

 

What are transposable elements (transposons)?

 

E.g. Transposable elements (transposons) are fragments of DNA that can move around in the genome and insert at target sites 4/3

 

 

If you looked at Drosophila and compared it to mammals, do you think it contains a lot of transposon derived genome?

 

No 4/3

 

 

Do you think a lot of new mutations in Drosophila are caused by mutations?

 

Yes indeed 4/3

 

 

What are Class II/Class I elements?

 

Elements that replicate via DNA (II DNA transposons) or RNA (I, or Retrotransposons) intermediates. 4/4

 

 

Give at least 3 examples of large DNA transposon families having terminal inverted repeats (the "normal" case) ?

 

Mutator, Merlin, PiggyBac, P, Transbib, PIF-Harbinger, hAT, TC1-Mariner, ... 4/9

 

 

Explain how terminal inverted repeated Class II transposons propagted (the molecular biology) a scheme would be helpful ?

 

See 4/12ff

 

 

When one looks at moving transposons of Class II, one can sometimes observe a footprint, what is that ?

 

After instertion of a DNA transposon the target site is duplicated, after excision this duplication remains 4/14

 

 

When one looks at moving transposons of Class II, one can sometimes observe a footprint, what is that ?

 

After instertion of a DNA transposon the target site is duplicated, after excision this duplication remains 4/14

 

 

When one looks at moving transposons of Class II, what is a precise excision ?

 

After excision of a transposon, homology directed repair of a transposon free site, restores the state before the transposon inserted, i.e no footprint 4/14

 

 

What are helitrons ?

 

Helitrons are Class II transpons, that seem to replicate via a rolling circle mechanism. In any case they are charcaterized by the RepHel protein 4/22ff

 

 

What is maverick?

 

An nodd transposon potentially related to DNA viruses 4/29

 

 

Are transopsons only bad for the host?

 

No some transposons have been domesticated, where the DNA binding domain is often reused (also they might contribute to plasticity of the genome) 4/32ff

 

 

Give three examples of LTR family transposons and give the class (i or II) they belong to?

 

Class I: Copia, Gypsy, Bel-Pao, Retrovirus, ERV 4/42

 

 

Draw a scheme how non LTR elements might replicate?

 

See 4/49

 

 

Lecture 5 small RNAs

What kind of small RNAs exist in bacteria?

 

Riboswitches, protein binding sRNAs,base-pairing sRNAs (cis-, trans-encoded); CRISPR RNAs

 

 

Explain the diference of cis- and trans-encoded base-pairing sRNAs.

 

cis-encoded: located on the antisense strand of the target RNA, perfect complementarity to target mRNA, act negatively (mRNA degradation, Transcription termination)

 

 

Draw a scheme of a CRISPR array.

 

See 5/9 , important features: DNA repeats, Unique spacers, CAS genes

 

 

How do CRISPR RNAs provide resistance against bacteriophages?

 

During phage infection, new spacers corresponding to sequences of this phage are integrated into existing CSIPR arrays.

 

 

What is the difference of miRNAs and siRNAs?

 

miRNAs: encoded in the genome by specific MIR genes; regulate developmental and physiological events; two Cleavage processes (Drosha, Dicer)

 

 

Draw a scheme of the generation of miRNAs.

 

See 5/20

 

 

What proteins are necessary for the generation of miRNA (with function)?

 

Drosha (cleaves pri(mary)-miRNA to pre-miRNA), Dicer (cleaves pre-miRNA to miRNA duplex), RISC complex (removal of one strand of miRNA, binding of complementary target mRNA, degradation of target mRNA)

 

 

What is the structure and function of ARGONAUTE proteins?

 

Structure: PAZ domain for recognition of 3‘ end of miRNA; RNAse domain for cleavage of target RNA

 

 

What are the differences between animal and plant miRNAs (4 examples)?

 

See 5/29

 

 

How does viral induced gene silencing work in plants?

 

See 5/36

 

 

Which plant-specific proteins are essential for RNA-directed DNA methylation and what is their role?

 

RNA-Polymerase IV (production of siRNA) and RNA-Polymerase V (Recruitment of ARGONAUTE to DNA)

 

 

What are piRNAs and what is their funtion?

 

PIWI-interacting RNAs; Transposon control in germ cells and stem cells in animals

 

 

Describe two applications of small RNA technique.

 

See 5/53-55

 

 

Lecture 6 Sequencing

Describe the principle of Sanger sequencing?

 

dideoxyy method, uses ddNTPs flouresncently labeled, extension stops, mix is run on gel in CE for a proper answer see 6A/4-14

 

 

What do you see in a sequence trace?

 

The intensities for the different dyes (translates to TCGA) at each position, from the peaks the sequence can be read 6A/14

 

 

What is a Phred score and is it linear?

 

A base quality score, no it is not linear, 10 units decrease the error rate by 10fold 6A/15

 

 

Describe the principle of Pyrosequencing?

 

Nucleotides are flowed in one after the other, upon incorporation PPi is released which is converted by Sulfurylase to ATP which is used by luciferase to produce light. Apyrase uses up unused nucleotides for a proper answer see 6A/17-25

 

 

What causes a problem for precise Pyrosequencing (i.e. where would you likely find sequencing errors)?

 

Homopolymers of the same Base, while the produced light gets more intense with more PPi released, it is difficult to distinguish longer stretches 6A/17-25

 

 

Which deoxynucleotides are used in Pyrosequencing and why?

 

dGTP, dTDP, dCTP, dATP-alpha S. The latter is used as it is not reognized by luciferase instead of ATP but can be used by the polymerase 6A/27

 

 

Describe the principle behind the Ion torrent sequencing?

 

Nucleotides are flowed in in a sequence, as they become incoporated H+ is released this is detected by sensors 6A/27

 

 

What causes a problem for precise Ion Torrent sequencing (i.e. where would you likely find sequencing errors)?

 

Homopolymers of the same base, while the produced H+ gets more intense with more bases incorporated in each cycle, it is difficult to distinguish longer stretches for a proper answer see 6A/34

 

 

Describe the principle behind Illumina sequencing (w.o. bridge amplification)?

 

Nculeotides are blocked and fluorescently labeled, all are flowed in at the same time, the cell is imaged, the block and label removed and a next cycle commences 6A/41

 

 

Name two ways to assemble a genome and describe them in briefly ?

 

Overlay/Layout/Consensus 6A/45-47 and kmer/de Bruijn based graph 6A/50-52

 

 

Show how the assembly of these two reads would proceed in a de Bruijn based assembler if the size of the used piece were 4 and 3 : ATCGAG, TATAAT, GAGTAT ?

 

only 4 shown, as this is easy to display ATCG=>TCGA=>CGAG TATA=>ATAA =>TAAT GAGT=>AGTA=>GTAT, three generates one component in the graph only

 

 

What is scaffolding; what is it good for and how is it often done?

 

Bringing contigs together, i.e. joining these by e.g. pieces of reads originating from two sides of a stretch of DNA where the middle is unknown, the unknown part is padded with Ns, this is done to get fewer and large pieces 6A/54ff

 

 

Explain how a genetic map is constructed?

 

genetic markers are testes for linkage, linkage can be expressed in cM to relate markers to each other. More detail from 6B 8-22 needs to be given

 

 

What is an RFLP marker and how is it usually detected?

 

Restriction Fragement length Polymorphism, a SNP causes the creation or deletion of a restriction site in the DNA. DNA can be cut with the restrction enzyme run on a gel, blotted and interrogated with a (radioactive) probe hybridzing to that stretch of DNA 6B/24

 

 

What is an SNP marker and how would you detect it?

 

Single nucleotide polymorpgism. E.g. using Illumina Golden gate (explain the assay!) 6B/29

 

 

What is a pyhsical map?

 

Where markers are assigned to physical location on the chromosome 6B/31

 

 

Explain restriction mapping and be prepared to interpret the results (constructing a physical map)?

 

Cut with different restriction enzymes either alone or in combination, determine the “restriction map” by combinatoric analysis 6B/33

 

 

How do you separate really large chunks of DNA?

 

pulsed field gel electrophoresis 6B/37

 

 

Explain optical mapping?

 

DNA is stained and immobilized (kind of streched) restriction enzymes added, the DNA shrinks a bit, gaps become visible these are visualized 6B/40

 

 

Explain STS tagging?

 

See 6B/45

 

 

Lecture 7 GWAS

What is GWAS?

 

Genome wide association mapping. In a population phenotypes are correlated to markers.... 7

 

 

Where can GWAS be used?

 

Usually relatively common allels with low penetrance/effect size 7

 

 

What is linkage disequelibrium?

 

LD is the “nonrandom association of alleles at different loci.” 7/16

 

 

What can be a pitfall when you study a large panel with GWAS?

 

Population structure 7/21

 

 

When you visualize genetic marker structure from a diverse geogrpahic region by PCA, what can you sometimes observe?

 

That the structure shown reflects the geographic location somewhat (TRICKY BONUS: Explain why this might be the case) 7/24

 

 

Do you think GWAS is successful?

 

Based on the number of publications, it probably is 7/32

 

 

Lecture 8 Proteomics/Interactomics

Why does it make sense to study protein accumlation?

 

posttranscriptional regulation, posttranslational modification, different splicing and polyA isoforms 8/3

 

 

When you compare data from proteomics and transcriptomics what can you say ?

 

Usually these do not complete coincide (but it is not alwayas as bad as in 8/4)

 

 

What is DIGE, how does it work and where is it used ?

 

Differential gel electrophoresis, it is used to compare protein amounts. Proteins are labelled by a fluorescent label and run on a 2D Gel 8/7

 

 

If you were to inject a protein mixture into an MS would you likely get many meaningful results out and why ?

 

No, the mixture would be too complex there would be overlapping Signals Due to Multiple Charged States & overlapping Signals Due to Natural Isotope Abundance 8/9

 

 

What is MALDI?

 

Matrix assisted Laser Desorption ionization. Analyte embedded in matrix a laser pulse (righ wavelength) is given which ionizes matrix+analytes 8/14

 

 

Which ion(s) will you always see in MALDI?

 

The ions originating from the matrix 8/14

 

 

Explain ESI?

 

Electrospray Ionization details from 8/15 needed a drawing will help

 

 

Explain TOF and quadrupoles very rougly?

 

Just some principle needed, e.g. quadropole has four rods, and selects based on m/z ratio ....

 

 

How would you obtain an MS/MS spectrum on a tripple quadrupole machine?

 

Quad 1 can be used to select an ion, Quad 2 is used as a collision cell, in quad 3 resulting fragement ions are scanned for 8/23

 

 

What would you often need for an MS^n and where can this be useful?

 

An ion trap based instrument useful in elucidating structure especially if MS/MS is not too meaningful 8/27

 

 

Why does one often need the genome before the proteome?

 

Several algorithms don't sequence peptides based on the ion but compare spectrum obtained to simulated spectra 8/31ff

 

 

Explain the SILAC workflow?

 

see 8/41 ff

 

 

How could you directly compare two conditions in proteomics (name at least 2)?

 

SILAC, iTRAQ, ICAT or digestion with H2 18O

 

 

Explain a Mass Western?

 

For a protein a heavy,stable ion labeled peptide is ordered and this is used to tune the instrument, afterwards the peptide of interest is injected with the syntghetic one and quantified 8/56

 

 

Lecture 9 Metabolomics

Why is metabolomics important?

 

one gets an information about the cellular components, potentially their physiological state etc

 

 

Explain EI ionization and where is it often used?

 

Electron impact, the molecule is bombarded with an electron and often "falls apart". In a GC-MS system one often finds EI 9/5

 

 

Explain EI ionization and where is it often used?

 

Electron impact, the molecule is bombarded with an electron and often "falls apart". In a GC-MS system one often finds EI 9/5