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Biomed year 2 sem 1 > CMB2000 - research skills > Flashcards

CMB2000 - research skills Flashcards

1
Q

Principally, what does PCR do?

A

Amplify DNA

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2
Q

DNA replication in prokaryotes

A
  1. DNA helicase unwinds DNA -> replication fork
  2. RNA primers bind template -> dsDNA
  3. polymerase joins nucleotides together
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3
Q

what happens on the 5’->3’ strand during DNA replication (DNA polymerase)

A

DNA pol can only run 3’->5’
leads to okasaki fragments on lagging strand
joined together later using ligase

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4
Q

role of sliding clamps

A

keeps polymerase in place on the DNA during eukaryotic DNA replication

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5
Q

Components needed for PCR

A

DNA template, Primers, Taq polymerase, dNTPs, MgCl2 (co-factor), 10x tris HCl buffer

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6
Q

Steps of PCR

A
  1. dsDNA is boiled at 95C -> ssDNA
  2. cooled to 55C to anneal primers
  3. heat back up to 72C, extend with polymerase and dNTPs
  4. repeat to amplify DNA between primer locations
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7
Q

How are PCR products detected?

A

stained with an intercalating gel (e.g. ethidium bromide - red under UV), and products are ran on agarose gel using electrophoerisis

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8
Q

How to do PCR if you start with RNA?

A

reverse transcriptase PCR converts RNA to cDNA first

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9
Q

SYBR green

A

fluorescent marker that binds to grooves of dsDNA

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10
Q

TAQMan

A

fluorescent, uses probes with reporter and quencher
more PCR product = more fluorescence

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11
Q

reference genes

A

housekeeping genes at a constant level of expression

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12
Q

Why is PCR clinically valuable?

A

Sensitive, specific, cheap, rapid, robust

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13
Q

3 uses of PCR in diagnosis/prognosis

A
  1. genotyping the patient
  2. genotyping the pathogen
  3. phenotyping the disease (Snapshot in time)
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14
Q

what is PCR-RFLP?

A

PCR Restriction fragment polymorphism
- uses restriction endonucleases

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15
Q

what is ARMS-PCR?

A

Amplification refractory mutation system
- detects allelic variants using allele specific primers

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16
Q

three conventional diagnostic techniques that can be replaced with PCR

A

microscopy, culture, patient antibody response

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17
Q

key tehnique for phenotyping a disease?

A

quantitative PCR

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18
Q

what is the ct value?

A

cycle threshold - how many cycles needed before fluorescence is visible.
lower ct = more nucleic acid/DNA

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19
Q

at what stage would you expect the lowest ct value?

A

immediately after infection, before immune system has had time to act

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20
Q

4 steps of DNA isolation

A
  1. cell lysis
  2. DNA purification from cell extraction
  3. concentrate DNA
  4. measure DNA purity and concentration
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21
Q

methods of cell lysis/DNA extraction

A

Biological - enzymes (lysozyme in bacteria, sappanin in eukaryotic)
physical - osmotic pressure/ freeze-thaw
mechanical - grinding, friction, shearing

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22
Q

DNA purification

A

Phenol-chloroform extraction
- then centrifuged
OR
column purification commercial kits
- silica membrane

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23
Q

Restriction endonucleases

A

molecular scissors that cut DNA in precise location

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24
Q

what are restriction endonucleases used for?

A
  • make recombinant DNA molecules
  • cut DNA insto defined fragments
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25
Q

Types of REs

A

sticky ends or blunt ends

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26
Q

RE recognition sites are often….

A

palindromic

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27
Q

Agarose gel electrophoreisis

A

separates DNA fragments
agarose = polysaccharide from seaweed

28
Q

principle of electrophoreisis

A
  • polymerised agarose is porus -> DNA can move along it
  • samples migrate along the gel according to size/shape/charge
  • smaller/compact molecules will move further
  • visualise with intercalating dyes
29
Q

what does CRISPR/Cas9 stand for?

A

CRISPR = Clustered Regulatory Interspaced Short Palindromic Repeats
Cas = CRISPR associated proteins

30
Q

3 components of CRISPR/Cas9 complex

A
  1. Cas9
  2. crRNA
  3. tracrRNA
31
Q

CRISPR as an adaptive immune regulator

A
  • invading DNA recognised and cut by Cas1-Cas2 -> fragments
  • protospacers integrated into CRISPR locus
  • viral reinfection -> transcription of protospacers -> bind Cas9
  • Cas9/RNA duplex recruited to invading DNA strand
  • Cas9 cuts DNA stands -> DSB -> prevents infection
32
Q

What does the Cas operon encode?

A

cas proteins required for DNA cleavage

33
Q

components of the CRISPR locus

A

transactivating RNA
Cas operons
identical repeat array
spacer of invading RNA (between tracrRNA and crRNA)

34
Q

Engineered CRISPR/Cas9 in the lab

A
  • composite gRNA made using linker loop
  • deposition of Cas9/gRNA at desired locus -> site-specific cleavage via nuclease activity
  • repair of DNA break by endogenous DNA repair pathways allows specific genome edits
35
Q

gRNA =

A

tracrRNA + crRNA

36
Q

two mechanisms of cellular DNA repair machinery

A
  1. Homology directed repair (HDR)
  2. Non-homologous end-joining (NHEJ)
37
Q

CRISPR-mediated gene knockout via NHEJ

A

Target Cas9-gRNA complex to gene of interest
DSB introduced
Cell repairs break via NHEJ (error prone)
Indels introduced -> frameshift -> premature stop-codons introduced
normal gene product not expressed

38
Q

CRISPR-mediated gene knockout via HDR

A

DSB introduced by Cas9-gRNA complex
Introduce a template that will be used to repair DSB by HDR
PAM sites are removed from HR template -> prevents re-targeting of the region
Inserts of several kb are possible

39
Q

PAM site

A

prospacer adjacent motifs

40
Q

Ex-vivo delivery of CRISPR in the clinic

A
  1. remove cells from the patient/donor
  2. edit genome
  3. screen and expand cell populations
  4. engraft cells back into patient
41
Q

in-vivo delivery of CRISPR in the clinic

A
  1. package CRISPR/Cas in a delivery vehicle
  2. Deliver to patient
42
Q

Steps to obtaining genomic sequences from an organism

A
  1. obtain organism’s genomic DNA
  2. break DNA into small fragments
  3. Obtain the DNA sequence from all the fragments
  4. search for overlaps to help reconstruct the sequence
  5. fill in the missing gaps in the genome sequence
43
Q

computer analysis of protein sequence

A
  1. predict function - role of model organism
  2. prediction of protein localisation
  3. prediciton of protein domains/modification
44
Q

What does BLAST search do?

A

identify conserved domains

45
Q

what does NetPhos search for?

A

potential serine/threonine/tyrosine phosphorylation sites

46
Q

uses of genome sequence within an organism

A

identification of regulatory sequences
characterisation of protein families

47
Q

regulatory sequences

A

identify all promotors containing a transcription factor binding binding site

48
Q

Functional genomics experiments…

A

describe gene functions and interactions

49
Q

microarrays measure…

A

hybridisation

50
Q

illumina sequencing

A

fragments of DNA (library) bound to solid surface (flow cell)
solid phase PCR forms clonal clusters
only one base can be added per cycle - modified nucleotides with fluorescent group that blocks extension
reversible termination allows sequencing to proceed to next cycle

51
Q

steps of RNA-Seq

A

poly A selection -> fragmentation
Random priming (to remove bias)
cDNA synthesis (RT)
End repair, phosphorylation and A-tailing
Adapter ligation, PCR amplification and sequencing

52
Q

general experimental schema in sequencing

A
  1. Enrich
  2. Sequence
  3. Analyse
53
Q

ChIP-Seq

A

Cross link proteins to DNA
Isolate and shear DNA
Immunoprecipitate protein of interest
reverse cross linking
purify DNA
sequence

54
Q

ATAC-Seq

A

Assay for Transposase-Accessible Chromatin
Relies on transposase Tn5 (highly active, efficiently cuts DNA and ligase adapters to ends)
Adapter ligated fragments isolated -> amplified -> sequenced

55
Q

bisulphite sequencing

A

Determines the methylation state of DNA
Methylated cytosine is protected from deamination
Unmethylated cytosine converted to thymine via uracil

56
Q

BS-Seq

A

Identifies sites of methylation - sequences bisulphite treated and untreated samples
quantitative estimates of methylation
identifies hypo- and hyper- (transcriptional silencing) methylated regions

57
Q

3 licences required for research on animals

A
  1. personal licence for the researcher
  2. project licence for the study
  3. establishment liscence for where study takes place
58
Q

standard transgenic mouse approach

A
  1. gene of interest injected into pro-nucleus of fertilised mouse egg
  2. injected eggs transferred to pseudo-pregnant recipient mouse
  3. all offspring are screened for expression of transgene by DNA analysis
59
Q

gene-targeted transgenic approach

A
  1. isogenic transgene with drug selection gene introduced to embryonic stem cell
  2. drug selection used- surviving cells screened for transgene integration
  3. correctly targeted cells are injected into mouse blastocytes
  4. blastocytes transferred to psuedo-pregnant mouse
    5, chimaeric offspring are identified and bred -> germline transmission of transgene
60
Q

transgenic mouse vector construct

A

tissue specific promoter - gene of interest - 3’ protein tag for detection, polyA tail

61
Q

Cre recombinase

A

catalyses site-specific recombination between two LoxP sites

62
Q

Flippase

A

catalyses site-specific recombination between two FRT sites

63
Q

which gene allows straight forward knockouts

A

loxP

64
Q

genes that allow conditional knock outs

A

FRT and LoxP

65
Q

cre-lox system for conditional alleles

A
  1. manipulation -> targeted, floxed allele in mouse
  2. second mouse is transgenic for cre recombinase
  3. mice are crossed -> mouse with cre and floxed alleles
  4. tissue specific deletion of the floxed allele in tissues w/ cre recombinase
  5. can also have inducible cre expression
66
Q

knock-in mouse

A

mostly used to introduce a human mutation into the mouse
uses CRISPR/Cas9

Biomed year 2 sem 1 (7 decks)

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