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Cell Biology and Genetics > Simple molecular techniques > Flashcards

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
Q

What are the requirements of PCR?

A

DNA fragment to be copied

Primers - forward and reverse

Free dNTPs

Taq polymerase

Thermocycler

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26
Q

What are primers?

A

Short single-stranded sections of DNA

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27
Q

What are the stages of PCR?

A

Denature DNA

Add primers

Elongation

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28
Q

How is DNA denatured in PCR?

A

Heating mixture to 95C
breaks hydrogen bonds between complementary base pairs
separating into single strands

29
Q

How are primers added in PCR?

A

Mixture cooled to temperature below 95C

allows primers to bind to DNA strand by complementary base pairing

30
Q

How does elongation occur in PCR?

A

Taq polymerase elongates DNA strand from primers

31
Q

What is a condition of the primers used in PCR?

A

Must have complementary base sequences to the ends of the DNA fragment to be copied

32
Q

What is a condition of Taq polymerase used in PCR?

A

Thermostable - doesn’t denature at high temperatures

33
Q

What does each PCR cycle produce?

A

Doubles the number of DNA fragments

giving exponential increase

34
Q

What is the purpose of the thermocycler?

A

Control temperature during each cycle of PCR

35
Q

What are some of the uses of gene cloning?

A

Discover functions of genes

Produce useful proteins

Gene therapy

36
Q

What are some of the uses of restriction analysis?

A

Investigate mutations

  • introduce or remove restriction sites
  • insertions, deletions give different sized DNA fragments
37
Q

What does protein gel electrophoresis separate proteins based on?

A

Size

Shape

Charge

38
Q

What does a darker band on gel electrophoresis results mean?

A

More DNA/protein in that band

39
Q

What is the purpose of SDS page?

A

Separate out proteins

40
Q

What does SDS-PAGE separate proteins based on?

A

Size only

41
Q

How does SDS-PAGE give proteins the same shape and charge?

A

Use chemicals to break bonds in secondary and tertiary structure, convert protein into linear shape

Use chemicals to add charges on to proteins

42
Q

What is the purpose if isoelectric focussing?

A

Separate out proteins

43
Q

What does isoelectric focussing separate proteins based on?

A

Charge only

44
Q

What are the requirements of isoelectric focussing?

A

Proteins to be separated

Gel with pH gradient across it

45
Q

Why do proteins move across the gel in isoelectric focussing?

A

Proteins attracted by positive/negative charges at one end of gel

46
Q

When do proteins stop moving across the gel in isoelectric focussing?

A

When reach pH equal to their pI
have no net charge
so stop moving

47
Q

What is the purpose of 2D-PAGE?

A

Separate out proteins

48
Q

What are the processes involved in 2D-PAGE?

A

Isoelectric focussing

SDS-PAGE

49
Q

What is an epitope?

A

Few amino acids on antigen that antibody binds to

50
Q

How many B lymphocytes produce polyclonal antibodies?

A

Many B lymphocytes

51
Q

How specific are polyclonal antibodies?

A

Specific to one antigen

but bind to multiple epitopes

52
Q

How many B lymphocytes produce monoclonal antibodies?

A

One B lymphocyte

53
Q

How specific are monocloncal antibodies?

A

Specific to one antigen

and one epitope

54
Q

What is the purpose of Western blotting?

A

To see if a specific protein is present in a mixture of proteins
and its properties

55
Q

What is the first step of Western blotting?

A

Banding pattern from protein gel electrophoresis moved to nitrocellulose paper

56
Q

What is added to the nitrocellulose paper in Western blotting?

A

Primary antibody

followed by secondary antibody

57
Q

What is the condition of the secondary antibody in Western blotting?

A

Enzyme linked

enzyme catalyses colour-changing reaction

58
Q

What is the purpose of the secondary antibody being enzyme-linked?

A

If see colour change
means secondary antibody bound to primary antibody, which bound to protein
so protein is present

59
Q

What information does the position of the secondary antibody on the nitrocellulose paper give about the protein?

A

It’s charge

and size

60
Q

What is the function of ELISA?

A

See whether a specific protein is present in a mixture of proteins
and to see it’s concentration

61
Q

How does ELISA work?

A

Similarly to Western blotting

except no previous protein gel electrophoresis, nitrocellulose paper etc.

62
Q

How does ELISA measure a protein’s concentration?

A

Amount of colour change is proportional to concentration of protein

63
Q

What do enzyme assays measure?

A

Enzyme activity

64
Q

What do antibodies bind to?

A

Epitope on antigen protein

65
Q

What are the uses of separating out proteins?

A

Separate out proteins in blood, tissues

66
Q

What are the uses of ELISA?

A

Measure concentration of proteins in the blood

raised/lowered could indicate disease state

67
Q

What are the uses of measuring enzyme activity?

A

Measure enzyme activity in tissues

raised/lowered could indicate disease state

68
Q

What are some examples of increased enzyme activity indicating disease state?

A

AST/ALT - liver disease

CK - myocardial infarction

Cell Biology and Genetics (32 decks)

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