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Simple molecular techniques Flashcards

1
Q

What are restriction enzymes?

A

Endonucleases that cut specific DNA sequences

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2
Q

What are the specific DNA sequences that restriction enzymes cut called?

A

Restriction sites

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3
Q

What are some common features of restriction sites?

A

Less than 10bp long

Palindromes

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4
Q

What are palindromes, in the context of DNA?

A

Both strands of DNA read the same base sequence in opposite directions

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5
Q

What are the types of cuts produced by restriction enzymes?

A

Blunt ends

Sticky ends

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6
Q

What is smeant by sticky ends?

A

Staggered cut

single-stranded overhangs

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7
Q

What is the purpose of DNA gel electrophoresis?

A

To separate out DNA fragments

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8
Q

DNA gel electrophoresis separates out DNA fragments based on their…?

A

Size

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9
Q

What are the requirements of DNA gel electrophoresis?

A

Gel

Buffer

Power supply

Staining

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10
Q

What is the purpose of the gel in DNA gel electrophoresis?

A

To give resistance to DNA fragments

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11
Q

What is the purpose of the buffer in DNA gel electrophoresis?

A

Maintain charge on DNA fragments

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12
Q

What is the purpose of the power supply in DNA gel electrophoresis?

A

Generate charge difference across gel

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13
Q

What is the purpose of staining in DNA gel electrophoresis?

A

So the DNA fragments are visible and can be identified

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14
Q

Why do the DNA fragments move across the gel in DNA gel electrophoresis?

A

DNA is negatively charged

will move towards the positive end of the gel

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15
Q

Why do DNA fragments separate out when they are moving across the gel in DNA gel electrophoresis?

A

DNA fragments are different sizes, weights
heavier DNA fragments face more resistance from gel
move slower across gel

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16
Q

How are the results of DNA gel electrophoresis interpreted?

A

Comparing position of unknown lengths of DNA

to positions of known lengths of DNA

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17
Q

What is gene cloning?

A

Producing copies of specific genes and their products

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18
Q

How are restriction enzymes used in gene cloning?

A

Used to isolate the gene from the rest of DNA

Used to cut the plasmid open

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19
Q

How are plasmids used in gene cloning?

A

Gene is inserted into plasmid

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20
Q

How is the specific gene inserted into the plasmid in gene cloning?

A

Because the gene sticky eneds are complementary to the plasmid sticky ends
base pairs form

DNA ligase anneals the two together

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21
Q

What is done to the recombinant plasmid in gene cloning?

A

Mixed with bacteria

some bacteria take it up - called transformation

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22
Q

What type of gene do plasmids commonly contain?

A

Antibiotic resistance genes

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23
Q

How are transformed bacteria told apart from non-transformed bacteria in gene cloning?

A

Only transformed bacteria will have specific antibiotic resistance gene
only these bacteria will survive in environment of that antibiotic

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24
Q

What is the purpose of PCR?

A

To amplify DNA fragments

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25
What are the requirements of PCR?
DNA fragment to be copied Primers - forward and reverse Free dNTPs Taq polymerase Thermocycler
26
What are primers?
Short single-stranded sections of DNA
27
What are the stages of PCR?
Denature DNA Add primers Elongation
28
How is DNA denatured in PCR?
Heating mixture to 95C breaks hydrogen bonds between complementary base pairs separating into single strands
29
How are primers added in PCR?
Mixture cooled to temperature below 95C | allows primers to bind to DNA strand by complementary base pairing
30
How does elongation occur in PCR?
Taq polymerase elongates DNA strand from primers
31
What is a condition of the primers used in PCR?
Must have complementary base sequences to the ends of the DNA fragment to be copied
32
What is a condition of Taq polymerase used in PCR?
Thermostable - doesn't denature at high temperatures
33
What does each PCR cycle produce?
Doubles the number of DNA fragments | giving exponential increase
34
What is the purpose of the thermocycler?
Control temperature during each cycle of PCR
35
What are some of the uses of gene cloning?
Discover functions of genes Produce useful proteins Gene therapy
36
What are some of the uses of restriction analysis?
Investigate mutations - introduce or remove restriction sites - insertions, deletions give different sized DNA fragments
37
What does protein gel electrophoresis separate proteins based on?
Size Shape Charge
38
What does a darker band on gel electrophoresis results mean?
More DNA/protein in that band
39
What is the purpose of SDS page?
Separate out proteins
40
What does SDS-PAGE separate proteins based on?
Size only
41
How does SDS-PAGE give proteins the same shape and charge?
Use chemicals to break bonds in secondary and tertiary structure, convert protein into linear shape Use chemicals to add charges on to proteins
42
What is the purpose if isoelectric focussing?
Separate out proteins
43
What does isoelectric focussing separate proteins based on?
Charge only
44
What are the requirements of isoelectric focussing?
Proteins to be separated Gel with pH gradient across it
45
Why do proteins move across the gel in isoelectric focussing?
Proteins attracted by positive/negative charges at one end of gel
46
When do proteins stop moving across the gel in isoelectric focussing?
When reach pH equal to their pI have no net charge so stop moving
47
What is the purpose of 2D-PAGE?
Separate out proteins
48
What are the processes involved in 2D-PAGE?
Isoelectric focussing SDS-PAGE
49
What is an epitope?
Few amino acids on antigen that antibody binds to
50
How many B lymphocytes produce polyclonal antibodies?
Many B lymphocytes
51
How specific are polyclonal antibodies?
Specific to one antigen | but bind to multiple epitopes
52
How many B lymphocytes produce monoclonal antibodies?
One B lymphocyte
53
How specific are monocloncal antibodies?
Specific to one antigen | and one epitope
54
What is the purpose of Western blotting?
To see if a specific protein is present in a mixture of proteins and its properties
55
What is the first step of Western blotting?
Banding pattern from protein gel electrophoresis moved to nitrocellulose paper
56
What is added to the nitrocellulose paper in Western blotting?
Primary antibody followed by secondary antibody
57
What is the condition of the secondary antibody in Western blotting?
Enzyme linked | enzyme catalyses colour-changing reaction
58
What is the purpose of the secondary antibody being enzyme-linked?
If see colour change means secondary antibody bound to primary antibody, which bound to protein so protein is present
59
What information does the position of the secondary antibody on the nitrocellulose paper give about the protein?
It's charge | and size
60
What is the function of ELISA?
See whether a specific protein is present in a mixture of proteins and to see it's concentration
61
How does ELISA work?
Similarly to Western blotting | except no previous protein gel electrophoresis, nitrocellulose paper etc.
62
How does ELISA measure a protein's concentration?
Amount of colour change is proportional to concentration of protein
63
What do enzyme assays measure?
Enzyme activity
64
What do antibodies bind to?
Epitope on antigen protein
65
What are the uses of separating out proteins?
Separate out proteins in blood, tissues
66
What are the uses of ELISA?
Measure concentration of proteins in the blood | raised/lowered could indicate disease state
67
What are the uses of measuring enzyme activity?
Measure enzyme activity in tissues | raised/lowered could indicate disease state
68
What are some examples of increased enzyme activity indicating disease state?
AST/ALT - liver disease CK - myocardial infarction

Cell Biology and Genetics (32 decks)

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