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Biology - Year 2 > Methods in Molecular Biology and Genetics > Flashcards

Methods in Molecular Biology and Genetics Flashcards

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1
Q

What factors affect the melting temperature (Tm) of DNA?

A

Length of probe/ target
GC content
Salt concentration

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2
Q

What conditions give rise to more stringent conditions in DNA hybridisation?

What does more stringency mean in terms of DNA base mismatches?

A

Higher temperature and lower salt concentration

DNA base mismatches are less likely to be tolerated under stringent conditions

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3
Q

What techniques involve hybridisation of nucleic acids?

A 
Southern blotting
FISH
Microarrays
Polymerase chain reaction
Sequencing
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4
Q

What is the order of the steps in PCR?

A

Denaturation (94 degrees C)
Annealing (60 degrees C)
Extension (72 degrees C)

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5
Q

Where does Taq polymerase start copying at?

What does it do?

A

DNA primers attached to the end of the desired gene

Makes complimentary DNA using DNA primer attached to desired gene as a starting point

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6
Q

Where do reverse transcriptase enzymes originate?

A

From RNA retroviruses

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7
Q

What is

  • an oligonucleotide
  • proofreading
  • an amoplicon
  • a template
A

Short synthetics nucleic acid
The ability of a polymerase to check and correct the accuracy of a new sequence
The amplified product of PCR
A single strand of nucleic acid used to direct synthesis of a complimentary strand

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8
Q

What do these reverse transcriptase approaches stick to:

  • gene specific primer
  • oligo dT
  • random primers
A

Only sticks to mRNA from your gene of interest

A primer made of ~20 T nucleotides, it will stick to all mRNAs by hybridising to the polyA tail

Often hexamers or otamers will stick to any RNA, whether it is mRNA, rRNA or tRNA

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9
Q

Name some experimental approaches that involve PCR.

A

Infectious disease diagnosis

Food quality

Environmental monitoring

DNA fingerprinting

Genetic disease diagnosis

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10
Q

Describe the features of these QPCR detection methods:

  • TaqMan
  • SYBR
A

Utilises a dual DNA probe
Enables multiplexing
Is highly specific

Involves the intercalation of a florescent dye
Does not discriminate between specific and non-specific amplification
Requires melting curve analysis

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11
Q

In QPCR what is the Ct value?

A

The cycle at which fluorescence from amplification crosses a threshold

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12
Q

What is the name given to samples of known concentration/ copy number used to calculate absolute concentrations from the Ct values of unknown samples?

A

Standards

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13
Q

What is a southern blot?

A

A technique that utilises probes to detect specific DNA sequences separated by electrophoresis

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14
Q

What is a nucleotide?

What is a nucleoside?

A

Has one or more phosphates

Has no phosphates

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15
Q

What is Taq polymerase?

What is the optimal temperature for Taq polymerase to work at?

What are the pros and cons?

A

A thermoaquatic polymerase

72 degrees C

Works rapidly, but has a relatively high error rate and not good proof reading (lacks endonucleases so can’t correct mistakes made)

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16
Q

What is a technical replica?

What is a biological replica?

Use examples

A

Measure mouse’s tail three times to get average

Measure a group of mice’s tails to get the average

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17
Q

What is a constitutive gene?

What is a facultative gene?

A

Housekeeping gene, expressed all the time

Expressed as and when needed

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18
Q

What is the process of a southern blot?

How is northern blotting similar/ different?

A

DNA molecules cleaved by restriction enzymes

Restriction fragments separated on an agarose gel

Blot onto membrane (under denaturing conditions)

Detect using complimentary strands: hybridisation with radioactively labelled DNA probe - get radio-autograph showing hybrid DNA

Northern blotting is similar but RNA is first denatured and remains denatured during electrophoresis

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19
Q

In microarray analysis what is the probe and what is the target?

How is a RNA sample labeled?

What are some of the applications of DNA microarrays?

A

DNA spots on the array are the probes, labeled cDNA is the target

Using CY3 or CY5 modified dNTPs

  • global analysis of gene expression
  • diagnostic and prognostic tool, e.g. different cancers
  • detection of deletions/ amplifications and other variations in genomes (use comparative genome hybridisation)
  • identification of transcription factor binding sites (use ChiP-on-chip analysis)
  • detection of DNA methylation patterns
  • identification of functional links between different sets of genes
  • identification of splice variants
  • identification of potential drug targets and pathways linked with disease progression
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20
Q

What does SDS-PAGE stand for?

What is SDS used for in gel electrophoresis?

What is western blotting?

A

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

To denature proteins before electrophoresis

Transfer to a membrane of protein separated using electrophoresis

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21
Q

What are two ways you can label antibodies (i.e. produced by polyclonal antibody production)?

What antibody are scientists most likely to label primary of secondary? What do they have affinity for?

A

Fluorescent labels
Horseradish peroxidase

Secondary antibodies, have strong affinity for primary antibody

22
Q

In proteomics what is immunocytochemisty/ immunohistochemistry (ICC/ IHC)?

A

Related to Western blotting in same way as in situ related to Northern blotting
Primary antibody applied to midroscopic slide containing cells (ICC) or tissue (IHC)
Visualised via secondary antibody using either fluorescent label or precipitation technique

23
Q

Describe two antibody independent methods for studying protein.

A

Two dimensional electrophoresis
First dimension isoelectrical focusing - separate proteins due to charge (determined by amino acid content) using a
Second dimension SDS-PAGE proteins separated according to size

MALDI-TOF

  • produces mass fingerprint unique to each protein
  • isolate protein and put in liquid solution, add proteolytic enzyme
  • vapourise protein with lazed, hurl towards detector
  • flight time proportional to ratio of mass and charge of protein
24
Q

Give examples of gene delivery systems.

A

Liposome mediated gene transfer

Retroviruses

Adenoviruses

Adeno-associated virus (AAV)

Lentivirus

25
Q

Describe RT-PCR and qPCR.

A

Reverse transcriptase PCR is used to qualitatively detect gene expression through the creation of complimentary cDNA from RNA

Real time PCR is used to quantitatively measure the amplification of DNA using fluorescent probes

26
Q

In a ChiP experiment what does sonicate do?

A

Fragments DNA

27
Q

What is transformation?

What is transfection?

A

Adding “naked” plasmids containing foreign DNA into bacterial cells

Adding “naked” plasmids containing foreign DNA into cultured mammalian cells

28
Q

Why do we have more proteins (300000 - 500000) than genes (21000)?

A

Protein modifications
Alternative promoters
Alternative splicing
Alternative polyadenylation sites

29
Q

What expression systems is pTriEx designed to work in?

A

Insect cell expression systems
E.coli expression systems
In vitro TnT system
Mammalian expression system

30
Q

In the Lac gene regulation system, what is the purpose of adding IPTG?

A

IPTG binds to the Lac repressor preventing it from binding to its recognition site in the DNA

31
Q

What features are required to express a protein in an in vitro TnT system?

A

T7 terminator
Kozak sequence
T7 promoter

32
Q

What are the pros of protein expression in an in vivo system?

What are the cons?

A

Protein will be folded correctly
Fast
Proteins will be post translationally modified

Small quantity
Expensive

33
Q

What techniques can tell us about the 3D structure of a protein?

A

NMR

X-Ray crystallography

34
Q

Which of the following methods can be used to detect in vivo DNA-protein interaction?
EMSA
ChiP
Foot-printing

A

ChiP and foot-printing

35
Q

Which of these are general features of reporter genes?
A) can often be detected by histochemical assays
B) indicate the presence of stress conditions
C) are all of bacterial origin
D) are used to delineate regualtory sequence elements
E) are used to characterise proteomes

A

A and D

36
Q

What property of cell physiology does the DNA foot printing assay examine most specifically?

A

The precise location on a segment of DNA where a transcription factor binds

37
Q

Put the stages of a typical ChiP experiment in order.

  1. Precipitation
  2. Sonicate chromatin
  3. Reverse cross linking
  4. Quantitative PCR
  5. Cross-link protein and DNA with formaldehyde
  6. Formation of immunocomplexes with antibody to transcribe factor of interest
A
  3. 1.
    3.
    4.
38
Q

What are the steps involved in generating transgenic mice by microinjection in order?

A
  1. Treat females with follicle stimulating hormone (FSH)
  2. Treat females with leutenising hormone (LH)
  3. Mate
  4. Isolate reproductive cells
  5. Microinject DNA into pronuceli
  6. Reintroduction into surrogate female
  7. Pups
39
Q

What are the benefits of generating transgenics by micro injection?
A) most animal species can be modified by this approach
B) all cells carry transgene
C) efficient use of animals
D) micro injection is technically very easy

A

A and B

40
Q

Are embryonic stem cells totipotent, multipotent, pluripotent or differentiated?

A

Pluripotent

41
Q

Where in a blastocyst are embryonic stem cells derived?

What happens to the other layer?

A

The inner cell mass of the (embryonic) blastocyst

The outer layer of the blastocyst goes on to generate the extra embryonic tissue (e.g. parts of the placenta and the membrane surrounding the developing embryo)

42
Q

In transgenesis what is a chimera?

A

An animal with a mixture of wild type and transgenic cells

When ES cells are reintroduced into the blastocyst of a developing embryo the resulting pups are a mixture of wild type and transgenic cells (this is a random mixture)

43
Q

What are the potential problems with non-targeted integration of a transgene?
A) can only be used to generate knock-outs
B) variable expression levels
C) disrupts the wild type version of the gene
D) essential gene distribution
E) integration into “silent” locus

A

B, D and E

44
Q

Of the following which relates to why choosing an appropriate promoter is important when developing a transgenic organism?
A) it directs where the DNA will be incorporated
B) it regulates the level and pattern of expression
C) it is not important because enhancers regulate gene expression
D) it promotes stable, reliable, genetic incorperation into the host

A

B

45
Q

Which of the following can influence the level and pattern of transgene expression?
A) of the location(s) of transgene insertion is random
B) if the number of transgene copies that integrate into the genome is random
C) if the transgene may be inserted into a region of transcriptionally silent DNA
D) all of the above

A

D

46
Q

In transgenesis what is the role of thymidine kinase?

A

Is an example of a negative selection marker. Allows for the selection of targeted, rather than random integration of the transgene. If transgene inserted it excludes TK gene. Works opposite way to neomycin resistance

47
Q

What is spatial gene inactivation?

What is temporal gene inactivation?

A

A knockout in a particular tissue

A knockout in a particular developmental stage

48
Q

True or false: during PCR there is always a doubling of product after each cycle? Why?

A

False

Not all primers hybridise 100% efficiently so nay give less than doubling. As PCR proceeds efficiency drops and amplification plateaus as resources are used up and product accumulates

48
Q

Taq polymerase starts copying at?
A) the end of free single stranded RNA
B) any open point
C) RNA primers attached to the end of a desired gene
D) DNA primers attached to the end of the desired gene

A

D

Taq polymerase makes complimentary DNA using DNA primer attached to the desired eggs at the starting point

48
Q

When DNA is heated primers anneal to DNA strands?

A

False, primers anneal when DNA is cooled

49
Q

In protein engineering what is

  • the origami phenotype
  • Rosetta phenotype
  • inducible expression
A

The origami phenotype involves mutations in txrB and gor genes to increase disulphide bond formation. This can help the folding of mammalian proteins in E.coli.

The Rosetta phenotype adjusts for differences in codon bias: these strains have been engineered to over-express tRNAs which are rare in bacteria but common in mammals.

The level of expression of a product can be controlled, particularly of its toxic, by using an inducible promoter.

50
Q

What is miRNA involved in?

A

Natural gene silencing mechanisms

Biology - Year 2 (9 decks)

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