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MCBG (Milly's) > Session 12+14 (Molecular Techniques) > Flashcards

Session 12+14 (Molecular Techniques) Flashcards

1
Q

What is needed for protein gel electrophoresis? (4)

A

Gel, buffer (maintains charge on sample), power, stain

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2
Q

What type of electrophoresis uses proteins intrinsic properties?

A

Serum protein electrophoresis

density of band = height of graph

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3
Q

What type of electrophoresis separates proteins according to size? How?

A

SDS-PAGE

Secondary and tertiary structures removed by beta-ME and SDS adds a negative charge to strand
Simply looking at polypeptides

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4
Q

What type of electrophoresis separates proteins according to charge? How?

A

IEF (isoelectrical focusing)

Gradient of pH is set up (low pH/+charge. High pH/-charge), protein moves until they reach a pH=pI then movement stops because they have no net charge

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5
Q

What type of protein gel electrophoresis separates proteins according to size and charge?

A

2D-PAGE

Charge (horizontal movement)
Size (vertical movement)

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6
Q

When would you use 2D PAGE?

A

When you have complex mixtures (ie whole cells)

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7
Q

What is the process of protein identification called?

A

Protenomics

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8
Q

What is an epitope?

A

A few amino acids on a protein that antibodies bind to

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9
Q

What are the two main types of antibodies?

A

Polyclonal - binds to many epitopes

Monoclonal- 1 epitope

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10
Q

When would you use antibodies as probes?

A

Western blotting

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11
Q

What does ELISA mean?

A

Enzyme linked immunoabsorbant assay

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12
Q

What are the 3 stages of PCR?

A

Denaturing
Annealing
Extending

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13
Q

What are the temperatures for the different stages of PCR?

A

Denaturing- 95
Annealing - 60
Plolymerising - 72

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14
Q

What builds the new strands on DNA in PCR?

A

DNA Taq polymerase

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15
Q

What sort of primers are needed for PCR?

A

Forward and reverse

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16
Q

What are the uses of PCR?

A

Amplify fragment
Investigate single base mutations
Investigate small deletion/insertions
Investigate Variation in genetic relationships

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17
Q

What enzymes cut DNA at specific palindromic sequences?

A

Restriction endonucleases

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18
Q

Why are DNA fragments negatively charged?

A

Phosphate group on nucleotides (PO4)

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19
Q

What is the positive electrode called?

A

Anode

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20
Q

What do you need for DNA gel electrophoresis? (4)

A

Gel
Buffer
Power supply
Stain

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21
Q

What are the uses of DNA gel electrophoresis? (3)

A

Investigate size of DNA fragments
Investigate mutations
Investigate DNA variation

22
Q

What is a plasmid + isolated gene to be copied called?

A

Recombinant plasmid

23
Q

What is the vector for cloning DNA usually?

A

A plasmid

24
Q

What is used to reseal plasmids?

A

Ligase enzymes

25
Q

Why clone human genes? (4)

A

Make useful proteins
Find out what genes do
Genetic screening
Gene therapy

26
Q

What do you use to get DNA from mRNA?

A

Reverse transcriptase

27
Q

What gene is isolated when making human insulin?

A

Proinsulin (immature form of the gene)

28
Q

Why can gel electrophoresis be used to separate molecules of different size?

A

Large DNA fragments move slowly, smaller fragments move faster

29
Q

What do restriction enzymes cut?

A

Phosphodiester bonds

30
Q

What is DNA sequencing?

A

Process of determining the precise order of nucleotides within a DNA molecule

31
Q

How is DNA sequencing done?

A

Heat DNA + primer + dideoxynucelotides (tagged)
Chains grow
Separate out new strands by gel electrophoresis
Use computers and laser to read

32
Q

Why are dideoxynucleotides used?

A

They lack an OH group meaning elongation is terminated

33
Q

In PCR, at what end does the primer attach and why?

A

The 3’ end because the DNA chain grows in a 5’-3’ direction

34
Q

What are allele specific primers?

A

Sequences that allow for a change in sequence and will bind accordingly

35
Q

What is southern blotting?

A

Separating DNA by gel electrophoresis
Transfer to nylon membrane
(Done by soaking gel in alkaline solution- creates ssDNA)
Mix with DNA probes (dont need 100% complementary)
Detection methods detect binding

36
Q

Why use southern blotting?

A

Allows for detection of difficult to find DNA
Can be used in conjunction with PCR to detect:
Gene structure expansion and repeats, mutations, genetic variation

37
Q

How is northern blotting different to southern blotting?

A

It uses RNA instead of DNA

38
Q

Why is northern blotting more difficult than southern blotting?

A

RNA degrades

39
Q

What sort of primer would you use for RT-PCR? Why?

A

One with many TTT’s @ 5’ end so it can bind to the polyA tail at the 3’ end of mature RNA

40
Q

What do you compare during microarray?

A

2 ‘conditions’

41
Q

How many DNA sequences do you look at at once in microarray?

A

Thousands

42
Q

What does microarray allow us to see?

A

Gene expression in an individual

43
Q

What is microarray?

A 
Putting all genes on plate,
Take someone's mRNA (from tissue)
Use RT to produce cDNA
Label cDNA
Wash cDNA over plate
cDNA will bind to genes
Therefore gene expression can be seen
44
Q

What are mini-satellites?

A

Repeating areas of base pairs
(Can have the same repeat multiple times at a single loci)
(Variable between homologous chromosome pairs)

45
Q

How does DNA fingerprinting work?

A

Use restriction enzymes to chop up DNA
Complete southern blotting
Compare runs

46
Q

What section of DNA are you looking at in DNA fingerprinting?

A

The non coding regions of the DNA

47
Q

What are the probes used for DNA fingerprinting?

A

Minisatellites

48
Q

What is karyotyping?

A

Lining up of chromosomes

49
Q

What does FISH stand for?

A

Fluorescent in situ hybridisation

50
Q

How do you do a FISH?

A

Make specific DNA sequence copy,
Label probe
Denature DNA and allow hybridisation
Section of DNA becomes visible

MCBG (Milly's) (15 decks)

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