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Flashcards in PCR and primer design Deck (92):
1

What is PCR?

A method for selectively amplifying a particular segment of DNA

2

What can PCR be thought of as?

A molecular photocopier

3

What can we do with a PCR product?

Digest with restriction enzymes
Sequence
Clone

4

Who invented PCR?

Kary Mullis

5

When was PCR invented?

In 1983

6

When was the first published account of PCR?

In 1985

7

In what year was the work on PCR awarded the Nobel Prize for Chemistry?

In 1993

8

How long should PCR primers be?

~20 bases long

9

What should the GC content of PCR primers be?

45-55%

10

What should the 3'-most base of a PCR primer ideally be?

G or C

11

What might a segment of DNA amplified by PCR represent?

A small part of a large and complex mixture of DNAs, e.g. a specific exon of a human gene

12

PCR is a photocopier capable of duplicating a part of a sentence or paragraph. What must there be locations for and what must be special about these locations?

Must be locations for the beginning and the end of the section to be copied that are identifiable
Locations must be unique to allow the copier to locate the correct piece of text

13

How long does it take PCR to amplify a usable amount of DNA (visible by gel electrophoresis)?

~2 hours

14

Template DNA for PCR needs to be highly purified, true or false?

False, can use a colony of bacteria or yeast

15

What is PCR powerful enough to amplify?

A single DNA molecule, e.g. from a single sperm cell

16

Prior to PCR, what did Southern blotting (1975) permit?

The rudimentary mapping of genes in unrelated individuals (RFLPs, insertions and deletions)

17

Prior to PCR, what did DNA sequencing (1978) require?

Genes to first be cloned into bacteria using vectors such as plasmids of lamda phage

18

Prior to PCR, what were the downsides to gene library construction and screening?

Could take many months and libraries had to be made for each individual genome analysed

19

Prior to Mullis, who invented the basic principle of replicating a piece of DNA using two primers in 1971?

Gobind Khorana

20

What was Khorana's principle of PCR limited by and how did Mullis improve on it?

Progress was limited by primer synthesis and polymerase purification issues
Mullis properly exploited amplification

21

How many cycles are there in a basic PCR reaction?

30-35 cycles

22

What steps do the PCR cycles comprise of?

Denaturation, 95 degrees C, 30 seconds
Annealing, 55-60 degrees C, 30 seconds
Extension, 72 degrees C, time depends on product size

23

What is needed for a basic PCR reaction mix?

Template DNA
Reaction buffer
Nucleotides (dNTPs)
Primers
DNA polymerase

24

What does a PCR reaction buffer typically contain?

Tris
Ammonium ions
Potassium ions
Magnesium ions
Bovine serum albumin
Detergents

25

What usually is the DNA polymerase used for PCR reactions?

Taq polymerase

26

At which cycle do target products begin to get made?

The third cycle

27

How many copes of DNA are there at 30 cycles?

~E9 target copies
60 other DNA copies whose length is undefined

28

The accumulation of target products in PCR is not strictly a doubling at each cycle in the early phase, true or false?

True

29

Increasing the cycle number above ~35 increases the amount of target product generated, true or false?

False, increasing the cycle number above ~35 has little positive effect

30

When does there become a plateau of target products?

When...
The reagents are depleted
The products re-anneal
The polymerase becomes damaged

31

What causes the accumulation of unwanted products in PCR?

When too many cycles are performed

32

Prior to thermal cyclers, how was cycling initially performed?

Using three water baths: one at each cycle temperature

33

When were thermal cyclers introduced?

1986

34

Why did early polymerases have to be replenished at each cycle?

Because they were not thermostable

35

What does Taq stand for?

Thermus aquaticus

36

When was Taq DNA polymerase first described?

1988

37

What does the 37 degree C temperature cause that results in unwanted products?

Non-specific priming

38

Describe PCR cyclers

Available from many suppliers
Many block formats & multi-block systems
Reactions in tubes, tube-strips, 96-well & 384-well microtitre plates

39

What happened to PCR and the use of Taq polymerase in PCR in the early days?

They were both patented

40

When did PCR patents expire in Europe?

On 28th March 2006

41

In the early days, fees were levied on enzyme and thermal cycler purchases, even for academic use, true or false?

True

42

When was the original Taq patent obatined by Cetus (now owned Roche) ruled invalid?

1st April 2003

43

What part of PCR may still be patented?

Engineered polymerases

44

What is the next step if PCR has not worked?

May need to optimise the reaction conditions

45

How can you check a sample using gel electrophoresis?

Is the product of correct size?
How many bands are present?
Is any product visible at all?

46

Outline the ways in which the PCR reaction can be optimised

Annealing temperature of the primers
Concentration of Mg2+ in the reaction
Reduce the amount of template DNA and polymerase - 'less is more'
The extension temperature (A/T rich region?)
The extension time
The denaturing and annealing times

47

What equipment can you use to optimise the annealing temperature?

A PCR cycler with a gradient function

48

How can you optimise the annealing temperature?

In temperature steps of 2 degrees C above and below

49

Primers have a calculated annealing temperature, but how must temperature be confirmed in order to optimise it?

Practically

50

When optimising the Mg2+ concentration in a PCR reaction, what can it sometimes be a compromise between?

Yield and specificity

51

What does the fidelity of PCR depend on?

Mg2+

52

When optimising the Mg2+ concentration, what is Mg2+ varied in steps of?

0.5mM

53

How much template DNA is sufficient for amplification of human genes?

Around 10ng

54

What should the amount of template DNA be reduced or increased to suit?

Genome complexity

55

If you are re-amplifying DNA, what should you do to the DNA first?

Dilute it extensively

56

What should you follow for the amount of enzyme?

The manufacturer's recommendations

57

If using concentrated enzyme, what should you do before setting up the PCR reactions?

Make a 'master mix'

58

What shouldn't you do when adding enzyme to a PCR reaction mix?

Adding more 'just to be certain'

59

Most PCR protocols recommend a long initial denaturation step of 5-10 minutes although this may not always be necessary. When will the longer step be needed?

If...
Using a hot-start enzyme that must be activated
The starting template is a bacterial cell suspension
The target DNA lies within a GC-rich region of DNA

60

Why do some PCR protocols recommend a 30 minutefinal extension step?

Because non-templated A base addition can leave the 3' ends 'ragged'

61

What is the disadvantage to 3' ragged ends in a PCR product?

Causes 'double' peaks when analysing PCR product length by capillary electrophoresis

62

Addition of the non-templated A is very inefficient, hence the need to allow lots of time in the final extension, true or false?

True

63

What does Taq DNA polymerase lack that is commonly present in some other polymerases, e.g. E. coli DNA polymerase I?

The 3' to 5' proof-reading activity

64

How often does Taq-misincorporate a base?

1 base in E4

65

What proportion of a 400 base pair target will contain errors after 20 cycles? Will the error distribution be evenly distributed or random?

33% of molecules
Error distribution will be random

66

Do errors in a PCR product matter? Why?

Yes if you want to clone the amplified DNA
An individual molecule may harbour several mutations
No, if you want to sequence the amplified DNA or cut it with restriction enzymes

67

How can you overcome errors in PCR products?

Use a proof-reading thermo-stable enzyme, such as Pfu, rather than Taq, or a mix of Pfu and Taq polymerases

68

What size range are amplification products typically?

100-1500 base pairs

69

What is the size of the PCR target limited by?

The integrity of the starting target DNA (less than 50kb)

70

Longer PCR targets are amplifiable (larger than 25kb), but what does it require?

Modified reaction buffer
Cocktails of polymerases
Longer extension times

71

Can RNA be amplified?

Not directly
The DNA polymerase requires DNA template and will not copy RNA

72

How can RNA be amplified?

mRNA can first be copied into cDNA using reverse transcriptase

73

cDNA is a template for PCR and it need not be double-stranded, true or false?

True

74

PCR products ought to ligate easily into a blunt-ended restriction enzyme site but there is a lower than expected efficiency. Why?

PCR products are not truly blunt-ended as Taq polymerase often adds a single non-templates base (usually A) to the 3' end

75

How can the addition of non-templated bases 3' of the PCR product be taken advantage of?

For TA cloning
Linearise the vector at a blunt-ended restriction enzyme site (e.g. EcoRV or SmaI)
Incubate the linear vector with Taq polymerase and dTTP to add non-templated Ts
Ligate together

76

When designing PCR primers, within how many degrees C should individual annealing temperatures be of one another?

Within 1 degrees C

77

When designing PCR primers, what must primers not base pair with?

Each other
With themselves
Form hairpins

78

When designing PCR primers, what must primers not anneal to?

Repetitive DNA elements, e.g. Alu elements in the human genome

79

Give an example of how a badly designed primer might form a hairpin

Self-complementary and be able to fold into a hairpin
The ' end of the primer is base-paired, preventing it annealing to the target DNA

80

Give an example of how a badly designed primer might form a dimer

Forms a dimer with itself or with the other primer

81

Primer dimers can be an excellent but unwanted substrate for the Taq polymerase, true or false?

True

82

It is widely recognised that the manual design of PCR primers is difficult and unreliable, so what have been devised which takes all of the design criteria into account?

Computer programs
Some are commercial, others free and open

83

What program at the Whitehead Institute for designing primers is probably the most reliable and versatile free tool currently available?

Primer3

84

What does Primer3 design primers on the basis of?

The DNA sequence entered

85

Why do we need to consider the whole genome and not just the segment of interest when designing PCR primers?

Because the primers might also amplify other segments whose sequence has not been taken into account

86

What does UCSC In-Silico PCR search?

The entire human genome looking for potential primer sites within 4000 bases of one another

87

What information does UCSC In-Silico PCR provide?

It performs a virtual PCR reaction if it finds other potential primer sites
Identifies the amplified region, its size and the primer melting temperatures
Provides a link to the genomic location

88

Are primer design tools linked to sequence variant databases?

No

89

What factors are there that might mean that only one allele of a DNA fragment gets amplified using PCR?

If a SNP occurs near the 3' end of a primer
If a long length polymorphism (e.g. a LINE1 insertion) occurs within the target region

90

Primer design is based on reference DNA sequences but what else should be considered?

Variants

91

What program can be used to check SNPs and other variants of a DNA sequence?

SNPCheck3

92

What does SNPCheck3 do?

Locates the primers in the genome
Performs a virtual PCR
Identified SNPs and variants under the primers
Identified SNPs and variants in the amplified region