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Medical Cell Biology & Genetics > Molecular Techniques > Flashcards

Molecular Techniques Flashcards

1
Q

What is a restriction enzyme?

A

An endonuclease produced by certain bacteria that cuts specific DNA sequences (restriction sites)

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2
Q

What do restriction sites usually contain?

A

Palindromes

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3
Q

What is a sticky end?

A

Where an endonuclease produces a staggered cut, leaving a few unpaired DNA nucleotides of one strand that extend beyond the other

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4
Q

What attribute does DNA gel electrophoresis separate fragments of DNA based on?

A

The size of the DNA fragment

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5
Q

In gel electrophoresis, what do DNA fragments move towards the positive or negative electrode? Why?

A

DNA fragments move towards the positive electrode, as they are negatively charged - DNA is negatively charged due to its phosphate groups

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6
Q

How will a small fragment of DNA move in comparison to a large fragment of DNA in gel electrophoresis?

A

A small fragment will move further, closer to the positive electrode, in comparison to a larger fragment

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7
Q

What is a plasmid? What specifically may a plasmid contain relevant to medicine?

A

A small circular double-stranded form of DNA found in bacteria - specifically, they may contain genes that infer antibiotic resistance

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8
Q

Describe the 4 basic steps of gene cloning.

A
  • isolate the relevant gene of interest using restriction endonucleases
  • insert this gene of interest into a plasmid vector, producing a recombinant DNA molecule
  • introduce this recombinant DNA to a suitable host cell
  • identify and clone the host cell containing the DNA of interest
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9
Q

Why would we clone human genes?

A
  • to produce a protein of interest on a mass level
  • find out what genes do
  • screen for certain disorders (genetic screening)
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10
Q

What holds complemtary nuclear acids in a DNA sequence together?

A

Hydrogen bonds

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11
Q

Why is a primer needed in PCR?

A

Taq polymerase can only extend a double-stranded DNA sequence, so a primer must be used to provide the double-strand

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12
Q

In PCR, what temperature is the DNA first heated to? Why is this?

A

The temperature is raised to around 95C - this breaks the hydrogen bonds that hold the 2 complementary strands of DNA together

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13
Q

In PCR, once the double strands have been separated, what happens next? What does this allow?

A

The temperature is cooled to room temperature - this allows the primers (needed for Taq polymerase to begin double-strand synthesis) to anneal to the single stranded DNA sequences

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14
Q

What is special about Taq polymerase?

A

It is able to withstand extremely high temperatures (such as this that are enough to break DNA strands apart) without denaturing

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15
Q

What type of DNA changes is PCR used to investigate?

A

To investigate small insertions/deletions within a gene

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16
Q

In PCR, are the primers known beforehand?

A

Yes, in order to amplify the specific sequence that you want to mass produce (you therefore must also know the sequence you want to copy)

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17
Q

What 3 attributes can proteins be separated on in protein gel electrophoresis?

A
  • size
  • shape
  • charge
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18
Q

How does protein gel electrophoresis separate proteins based on their size?

A

Larger molecules won’t pass through the viscous gel as quickly as smaller molecules will

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19
Q

What is SDS-PAGE short for?

A

Sodium dodecyl sulphate polyacrylamide electrophoresis

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20
Q

What does sodium dodecyl sulphate (SDS) do?

A

SDS breaks down disulphide bonds, breaking a proteins secondary and tertiary structure and leaving only its primary structure (i.e. a linear polypeptide chain) - SDS also adds negative charge to this linear polypeptide chain

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21
Q

What does SDS-PAGE separate proteins based on?

A

It separates different proteins on the basis of their size

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22
Q

What does isoelectric focusing separate proteins based on?

A

It separates proteins based on their charge

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23
Q

Briefly explain how isoelectric focusing works.

A

A stable pH gradient is established within a gel after the application of an electric field - different proteins are added, and migrate to the point where they each a pH equal to their isoelectric point - the gel is then stained to show where the proteins are distributed

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24
Q

In isoelectric focusing, why do proteins stop migrating once they have reached the pH where they are at their isoelectric charge?

A

They have no net charge at their isoelectric point, and so will have no net charge at this pH, so will stop migrating

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25
Q

Briefly describe how 2D-PAGE works.

A

2D-PAGE firstly separates proteins by their isoelectric point - having done this it then separates proteins based on their size

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26
Q

When is 2D-PAGE particularly useful?

A

When diagnosing disease states in different tissues

27
Q

What is proteomics?

A

The analysis of all proteins expressed in a genome

28
Q

At which residues does trypsin cleave a protein?

A

Lysine & arginine

29
Q

At which residues does chymotrypsin cleave a protein?

A

Tyrosine, phenylalanine, leucine, & tryptophan

30
Q

How are polyclonal antibodies produced?

A

An antigen is first injected into an animal to initiate an immune response - after a series of time the animal should have developed antibodies against the antigen - blood is then extracted from the animal and purified to obtain the antibody

31
Q

How are monoclonal antibodies produced?

A

Ask l8r

32
Q

Describe how a western blot is performed.

A

Firstly, proteins are separated by their size (usually via SDS-PAGE) - these proteins are then transferred to a nitrocellulose membrane where they are stained with a primary antibody specific to the target protein - the membrane is rinsed to remove unbound primary antibody and secondary antibody is added to the membrane - this binds the primary antibody, and is normally attached to a bioluminescent molecule or enzyme used to amplify the signal

33
Q

Describe how ELISA is performed.

A

Antigens from a sample are attached to a surface of a well - a specific primary antibody is added which binds the antigen - a secondary antibody is added to the well which is bioconjugated to an enzyme - the enzymes substrate is added, which will usually produce a marked change in the colour of the solution - the greater the change of colour, the greater the amount of antigen in the initial solution

34
Q

Overall, what does ELISA show?

A

ELISA identifies the presence of a particular substance, or the concentrations of the substance (usually protein) in solution

35
Q

What is the Vo?

A

The initial rate of reaction

36
Q

What is a continuous assay? Give 2 examples.

A

Where only one assay is needed to get a measure of the enzyme activity - examples include:

  • spectrophotometry
  • chemoluminesence
37
Q

What is an enzyme assay? Why are they important diagnostically?

A

A method measuring the activity of an enzyme - they are important diagnostically as they provide a measure of metabolic function of an enzyme

38
Q

What is a discontinuous assay? Give 2 examples.

A

Where several assays/samples are taken from an enzymatic reaction at several intervals to measure enzymatic activity - examples include:

  • radioactivity
  • chromatography
39
Q

What 2 methods can you use to denature a double-stranded portion of DNA? Specifically, what do these methods do?

A

By heating them or subjecting them to an alkaline pH - both of these break the hydrogen bonds connecting the double stranded DNA

40
Q

What does a Southern blot detect?

A

A southern blot is used to detect complementary DNA sequences (1 strand from the original DNA and 1 radioactive probe)

41
Q

What does a southern blot show?

A

The presence or absence of a particular DNA sequence, by using a radioactive probe

42
Q

In southern blotting, why is a DNA probe used?

A

After the DNA is cleaved by a restriction enzyme and then run over gel electrophoresis, the quantity of DNA, and the variation in different sizes, means all that is visible is a huge smear - a probe allows you to specifically identify the sequence of DNA you are probing for

43
Q

Describe the process of a southern blot.

A

Firstly, DNA fragments are run over an electrophoresis gel (smaller molecules will move further than larger DNA fragments) - the gel is soaked in an alkaline solution which denatures the DNA into single fragments - these are then transferred onto a nylon or nitrocellulose membrane - the filter is placed into a solution containing the labelled DNA probe, which will bind complementary single-stranded DNA on the filter - afterwards the filter is washed to remove any unbound probe and photographic film is used to detect binding

44
Q

Why is southern blotting used?

A

In order to analyse a gene for large duplications, repeats, or deletions, and to investigate for mutations or variation

45
Q

In southern blotting, do probes have to be 100% complementary?

A

No - even with 80% homology a DNA probe will bind, albeit less tightly

46
Q

In southern blotting, do probes affect the position of a target sequence on a gel?

A

No, they have no effect

47
Q

What is the best molecular technique to use to figure out a DNA nucleotide sequence?

A

The Sanger chain termination method

48
Q

What specific molecule allows identification of the DNA sequence in the Sanger chain termination method?

A

Dideoxynucleotide triphosphate - this has a H at the 3’ position as opposed to a OH- - this means that when inserted into a sequence, synthesis will end prematurely as a phosphodiesterase bond linking the next deoxynucleotide triphosphate in sequence will not be able to be formed, blocking elongation

49
Q

On a single strand of DNA, how are subsequent deoxynucleotide triphosphate molecules added?

A

Through the formation of a phosphodiesterase bond between the 5’ phosphate group and the 3’ hydroxyl group on the growing chain

50
Q

Describe the process of the Sanger method.

A

4 seperate tubes, each with a unique dideoxynucleotide triphosphate, are incubated with the target sequence - once incubated, these are run on a gel to determine the order of the nucleotide sequence in the DNA strand - in reality, nowadays radioactive dideoxynucleotide triphosphates are used in one tube and ran over a gel - the sequence is then determined by the next radioactive didepxynucleotide triphosphate that has halted elongation

51
Q

What can a northern blot detect?

A

How much mRNA is expressed in a certain tissue

52
Q

What feature of mRNA does reverse transcriptase PCR utilise? How?

A

The polyA tail - a 5’ primer with abundant thymine will bind the 3’ polyA tail, and reverse transcriptase will then be able to copy the mRNA

53
Q

What enzyme is used to separate the double-stranded RNA?

A

An RNAase

54
Q

What does each well in a microarray consist of?

A

Known sequences of DNA

55
Q

What can microarrays give?

A

A comparison of 2 conditions, based on the difference in colour of the wells of the microarrays

56
Q

Describe how a microarray works?

A

Reverse transcriptase PCR is used to make cDNA from mRNA - the cDNA can then undergo PCR and be amplified - from here, cDNA from 2 different conditions (eg wild type and disease-state) can be separately labelled - these can be mixed and hybridised to a microarray - depending which DNA sequences fluoresce or not, and comparing the levels of fluorescence of the colours you can see the difference in the composition of the DNA across the 2 conditions

57
Q

When Karyotyping a selection of chromosomes, what stage of nuclear division was the cell they were taken from at?

A

Metaphase

58
Q

What is Karyotyping?

A

Stain chromosomes, and pair them using their stains as markers for a homologous pair - this can then be used to compare deletions on a chromosomal level

59
Q

Describe the stages of DNA fingerprinting.

A

DNA is first extracted, then amplified using PCR - a southern blot is then performed, running the DNA fragments down an electrophoresis gel and transferring them to a nylon or nitrocellulose filter for hybridisation with a radio labelled probe molecule - this can then be compared to another DNA fingerprint to identify differences (if any) between the 2 DNA fingerprints

60
Q

In DNA fingerprinting, what specific sequences of DNA tend to be looked at? Why?

A

Usually short-tandem repeats are analysed - as they reside in non-coding regions (introns) they show great variability between individuals who aren’t related, and less variability between individuals who are closely related

61
Q

Why may a short-tandem repeat show great variability?

A

They are apparent in introns and so are not greatly conserved - therefore, mutations may occur here that have no effect on an individual’s viability so can persist - related individuals however tend to have less variation as they have directly received these short tandem repeats from family members

62
Q

What type of abnormalities does FISH (fluorescence in situ hybridisation) identify?

A

Chromosomal abnormalities

63
Q

What does FISH (fluorescence in situ hybridisation) help identify?

A

It allows you to identify where specifically on a chromosome a gene resides, and whether there are nay abnormalities within this gene across other chromosomes/samples

64
Q

What stage of replication are chromosomes analysed for FISH (fluorescence in situ hybridisation) taken?

A

Metaphase (or interphase)

Medical Cell Biology & Genetics (11 decks)

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