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Research Approaches > Midterm 2 > Flashcards

Midterm 2 Flashcards

1
Q

Recombinant DNA has had genes _____ or ______.

A

added; deleted

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2
Q

What are industrial, clinical, and research applications for recombinant DNA?

A
  1. Gene and hormone replacement therapy
  2. Identify disease + investigate SNPs for rare diseases + therapy
  3. Analyze gene + protein function. Understand tissue specific expression.
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3
Q

Cloning vectors are generally propagated in ______.

A

Plasmids because of their large insertion sites. Other vectors are used with bigger sites to create libraries.

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4
Q

How are bacteria made competent for recombinant introduction?

A

Heat or electrical shock

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5
Q

What are the two antibiotic resistance genes needed in plasmid vectors?

A

Tetracyline and Ampliciline. This helps with clonal selection, you know the recombinants are the ones that don’t grow.

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6
Q

What chemicals are used for blue-white colonal cultures?

A

X-Gal and IPTG

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7
Q

What are mini, midi, and maxi kits used for?

A

Recombinant DNA amplification.

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8
Q

What are 3 methods for transfection (delivery of recombinants into eukaryotic cells) to cause transduction?

A

Electroporation (shock), lipofection, and viral vectors.

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9
Q

What are the components of viral DNA?

A

Gag (viral core), Pol (transcriptase), Env, and VSV-G (envelope protein).

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10
Q

How is mRNA stabilized to form libraries?

A

Converted to cDNA with reverse transcriptase. Good to study production of new proteins or to determine specific expression and timing patterns.

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11
Q

How has the price of high speed sequencing changed?

A

From $50,000 to under $1,000.

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12
Q

What are the four research areas of sequencing?

A

Genomique (structure, fonction, evolution, mapping), Transcriptomatique (analyse ARNm,t,s, non-codants a grand echelle), Epigenomique, et Interactomique (proteine-ADN)

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13
Q

Describe genomic sequencing process.

A

Selective incorporation of ddNTPs creates chain-terminating triphosphates in vitro. Advantage? No bacterial culture or library.

Molecules are fused to adaptor sequences, attached to flowwell (puce de sequencage). Ensuite amplifies par PCR pour generer clusters d’ADNc with fluorescents added for live identification.

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14
Q

Why is a sur-echantillonnage massif utilise pour assembly?

A

Assurer que tout le genome est sequence + limiter d’erreur.

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15
Q

______ est liee a la qualite de sequencage.

A

Couverture

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16
Q

______ is avg number of a base sequenced divided by length in the reference gene.

A

Profondeur. Deep est 1000x. Ultra deep est 100 000x.

17
Q

______ est la pourcentage des bases lu at a certain depth. Varies across the gene.

A

L’entendue (breadth)

18
Q

What are the 3 steps of annotation in genomic sequencing?

A

Structural: Identification of coding regions. ID of ORFs, regulators, etc.

Functional: Information biologiques, interactions entre genes et niveau d’expression selon tissu/cell type

19
Q

What’s the most simple tool for identifying gene similarities based on homology?

A

BLAST

20
Q

ORA determines statistically % of genes in a pathway which _____ at a significance of ____.

A

Show a change in expression; p<0.05.

21
Q

Definez le mot “ontology”

A

Representation formelle d’un domain d’etude.

22
Q

How are geneontology categories presented?

A

As a tree (Directed Acylic Graph) Rouge is enrichissement negatif vs controle. Bleu est positif.

23
Q

Name 3 limitations of ORA.

A

Not all biological processes are well understood. Significance value is random, could be exclusionary. Ignores magnitude.

24
Q

What does Functional Class Scoring do?

A

Looks at large and small changes in gene expression. Calculates statistics for each gene and combines to determine unique value for a pathway. Permutation tests verify significance.

25
Q

What does GSEA calculate?

A

Enrichment Score (sum of Fold-Change) and ranks by expression.

26
Q

How do you analyze GSEA?

A

Deviation maximale (leading edge) represente le ES. Magnitude est en lien avec phenotype.

27
Q

Name 3 ways proteins and DNA interact.

A

DNA packaging, gene regulation, and DNA reparation

28
Q

What is Electrophoretic Mobility Shift Assay (EMSA)?

A

Studies linkage of gene on a nucleotide probe. Tests interaction affinity.

Can be pure protein sample or mixed. In agarose gel, the complex moves slowly. En ajoutant un anticorps specifique a la proteine on observe a supershift which identifies which proteins interact.

Can examine Kd affinity and number of binds between protein + sequence.

29
Q

What is DNAase footprinting?

A

DNA is protected by protein. So we mark DNA with P32 (marker) then expose to DNAase with and without protein mix (recombinant/nuclear)

30
Q

What is Pull-down d’ADN biothinyle?

A

Sequence is tagged with high affinity molecule like biotin, added to sample. DNA will like to proteins like transcription factors. Complex is precipitated with agarose/magnetic beads. Western blot.

31
Q

What does Chromatin Immuno-precipitation (CHIP) do?

A

Determines if proteins have specific place in genome. Localisation of histone modifications to identify epigenetic regulation.

32
Q

How does CHIP work?

A

Limits chromatin cross-linking, breaks down ADN-Chromatine-Proteine complexes to 500 pb fragments w/ sonication or endonucleases. Then, immunoprecipitation with specific antibodies.

Sequence the fragments.

33
Q

What is the use for Extinction genique?

A

View the effects of partial silencing rather than full knockout

34
Q

How does inhibiting les ARN inferentiels silence a gene?

A

Limits transposon proliferation and translation regulation via microRNA.

35
Q

What is the shape of microRNA?

A

Pinhead

36
Q

How are RNAi’s used in extinction genique?

A

miRNA is cleaved to 21 pb. siRNA and miRNA are integrated within RISC protein which directs silencing.

siRNA is easy to use and synthesize and causes temporary silencing. shRNA allows for longer-lasting effect (months) and less is required, which limits side effects.

Research Approaches (3 decks)

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