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Molecular Biology > Molecular Biology Week 2. > Flashcards

Molecular Biology Week 2. Flashcards

1
Q

Name the five steps involved in DNA isolation.

A 
Release
Disrupt and prep cell free extract
Remove Debris from extract
Purify DNA from extract
Measure quantity and quality of DNA.
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2
Q

What is an example of a specimen with nucleated cells?

A

A common example is WBCs from blood or bone marrow.

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3
Q

Name the long labour intensive method used to separate WBC from the plasma and other components and cells?

A

Differential Density gradient centrifugation. Ficoll Gradient.

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4
Q

Name the more efficient method of separating WBC from plasma and the other components and cells?

A

Differential Lysis which uses the osmotic fragility of the cells.
Hypotonic buffer = lysis of RBCs before WBCs. Centrifugation… pellet of WBCs.

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5
Q

What state are tissue samples in before DNA isolation?

A

Fresh or frozen tissues must be dissociated (separated)

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6
Q

Name the three techniques of dissociating a tissue sample.

A

Grinding frozen in liquid nitrogen.
Homogenising tissue.
Mincing using a scalpel.

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7
Q

How are fixed embedded tissues deparaffinated?

A

Through using xylene or less toxic xylene alternatives. Then tissue must be rehydrated.

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8
Q

What can be prepared from the released cells of a tissue sample?

A

Free cell extract, cells must be lysed.

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9
Q

What do some bacteria and fungi have in common that must be broken down before a cell extract is obtained?

A

Tough cell walls.

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10
Q

Name the 4 steps involved in obtaining a cell extract from a microorganism sample.

A

Culture is grown and harvested.
Cells are removed and broken down to produce extract
DNA is purified.
DNA is concentrated.

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11
Q

Name the three conditions which determine cells become a cell free extract.

A

The cell type (prokaryotic or eukaryotic)
Presence of a cell wall
Composition of the wall.

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12
Q

Name 4 methods involved in preparing cells to become a cell free extract/methods of cell wall and membrane disruption.

A

Naturally using a gentle buffer.
Enzymes
Grinding or vigorously mixing with beads/magnets
Treatment with detergents and bases such as NaOH

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13
Q

Name three methods of purifying cell free extract to becoming purified DNA.

A

Organic
Inorganic
Solid Phase Extraction

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14
Q

When purifying DNA which two methods are used in both inorganic and organic techniques. Which one method is unique to inorganic technique?

A

High salt
Low pH
Inorganic; Mix of chloroform and phenol

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15
Q

What does high salt, low pH and a mix of chloro and phenol do to the contaminants of DNA

A

Dissolves the hydrophobic contaminants such as lipids and lipoproteins and collects cell debris and strips away most DNA associated proteins.

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16
Q

What is used to degrade RNA?

A

RNase.

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17
Q

What does chloroform and phenol develop?

A

Develops a biphasic layer emulsion. After centrifugation; hydrophobic layer at bottom, hydrophilic on top with a white interface in the middle.

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18
Q

What is the white interface in the middle of the biphasic emulsion made up of?

A

Amphipathic components. Coagulated proteins.

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19
Q

What charge is DNA and RNA and what is its nature towards water?

A

DNA and RNA is hydrophilic and is negatively charged.

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20
Q

What happens to the test tube with the biphasic layer and the white interface of amphiphathic cells?

A

Upper hydrophilic layer is collected, DNa is precipitates using ethanol and a high conc of salt.
The Precipitate is collected, rinsed to remove the salt.
DNA is then dissolved in a buffer.

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21
Q

What is the safety issue with phenol?

A

“salting out” can be dangerous.

22
Q

How does the inorganic isolation method differ from the organic method in terms of separating the DNA in the solution.

A

Use of low pH and high salt to selectively precipitate proteins, separating DNA in the solution. Sodium Acetate.

23
Q

How is DNA collected after being separated in the solution?

A

It is precipitated in alcohol and then is pelleted and resuspended in buffer.

24
Q

What is used in solid phase isolation which is different to the two other methods?

A

DNA extraction is done using solid matrices.

25
Q

Name the steps of DNA extraction. Solid Phase Extraction.

A

Sample, lysis (release of nucleic acids) , binding (high salt buffer), washing (buffer), elution (water or buffer) and finally purification of nucleic acids.

26
Q

How are most solid matrices obtained in a laboratory environment?

A

Commercial kits in columns of different sizes.

27
Q

Why is it difficult to work with RNA in the lab?

A

Due to the widespread nature of RNases. These enzymes can also renature after autoclaving.

28
Q

Which is the RNA which is most sought after for gene sequencing?

A

mRNA. Which is 2.5 - 5 % of RNA.

29
Q

How are the cells lysed when doing an RNA extraction?

A

This step is done in a detergent or phenol in the presence of high salt or RNase inhibitors. DNase can be added also to remove any contaminating DNA.

30
Q

What is an example of a detergent which is a strong denaturant in which RNAses can be used?

A

Guanidine Thiocyanate.

31
Q

What is the ratio and the chemicals that are used to efficiently extract RNA?

A

Acid Phenol:chloroform:isoamylalcohol (25:24:1)

32
Q

How is messenger RNA (mRNA) isolated?

A

These are required to be isolated in many cases of making mRNA molecules. The polyA tails of the mRNaA molecules are exploited to isolate these molecules.

33
Q

How are the polyA tails exploited to isolate the mRNA?

A

PolyT and polyU oligomers bind to the poyA tail founr exclusively on the mRNA. Oligomers of thymine or uracil are immobilised on a matrix of resin column.

34
Q

What is done after washing away the other RNA molecules?

A

polyA mRNA is eluted on washing the column with a low salt buffer containing detergent.

35
Q

How is DNA stored?

A

As DNa is stable for long periods of time, it can be isolated in a purified DNA (stable at 4C) way or a non-purified (frozen in single use aliquots) DNA way.

36
Q

What are the three ways in which nucleic acids should be quantified?

A
  • ensuring that extraction was effective.
  • ensuring the stndardisation in further analysis of the DNA/RNA
  • determining the concentration of nucleic acids relative to the conco of protein that remains in the extracted sample.
37
Q

Name two methods in which nucleic acids are quantified?

A

Electrophoresis and spectrophotometric analysis.

38
Q

What types of applications is gel electrophoresis used for?

A

To analyse PCR (polymerase chain reaction) products, examine restriction digest patterns etc.

39
Q

What is Gel Electrophoresis?

A

It is the movement of charged molecules in an electric field. In which neg molecules move towards the pos electrode and pos molecules head towards the neg electrode.

40
Q

What are the two types of gel used in molecular biology regarding gel electrophoresis?

A

Agarose gels and polyacylamide gels.

41
Q

What is agarose?

A

It is a polysaccharide that forms gels with pores that vary in diameter depending on the conc of the agarose in the gel.

42
Q

What determines the range of DNA fragments that can be separated?

A

The gel concentration. Higher concentration = smaller DNA fragments can be separated.

43
Q

What are the brief steps to preparing the agarose gel in electrophoresis?

A
  1. Mix the agarose powder in a buffer soltion.
  2. Heat to dissolve, pour molten gel onto perspex plate and tape sides.
  3. Place comb to form wells for samples.
  4. Allow to solidify
  5. 1/2 dyes of known migration rate are added to the DNA before loading.
  6. Bands of DNA are visualised by using an ethidium bromide/sybr green staining either during or after electrophoresis.
44
Q

What is ethidium bromide?

A

This is a fluorescent molecule that intercalates between bases.

45
Q

How is DNA/RNa analysed using electrophoresis?

A

It can be analysed by resolving an aliquot of the sample on an agarose gel and generally staining with a fluorescent dye for visual inspection.

46
Q

The higher the molecular weight genomic DNA = ?

A

Shows a bright band with low mobility at the top of the gel.

47
Q

RNA shows up as = ?

A

two distinct bands of rRNA. quality and ratio

48
Q

How can DNA be quantiated in gel electrophoresis?

A

By the comparison of the fluorscent intensity of the sample with controls by v.i and or by densitometry.

49
Q

At what absorbancy does nucleic acid take in light at through adenine residues?

A

At 260nm.

50
Q

What does spectrophotometry do?

A

It indicates the quality of the nucleic acid. For good quality DNA: 1.6 - 2 x Abs @ 280nm.

51
Q

Name a development in spectrophometry which allows for fast and reliable DNA and RNA analysis?

A

Nanodrop technology.

Molecular Biology (8 decks)

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