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molecular genetics > exam 1 > Flashcards

exam 1 Flashcards

1
Q

What is the ideal primer length? What is the limit?

A

Ideal 18-24bp, can go up to 42

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2
Q

What is the ideal GC content of primers? What if it is not in those bounds?

A

Ideal 40-60%. Increase means higher temp for denature and secondary primer structures wont denature, lower means less stability

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3
Q

What is the difference of annealing temperature and melting temperature?

A

2-3C lower

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4
Q

What occurs during the melting temperature for primers?

A

Temperature where 1/2 old the DNA duplex dissociates and becomes single stranded

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5
Q

What is the ideal melting temperate range?

A

52-65C

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6
Q

What is the concern of primer secondary structures?

A

Can lead to poor or no yield of product/application fails

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7
Q

What are the common primer secondary structures?

A

Hairpins, self-dimer primer, heterodimer

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8
Q

What is the max number of repeats in primers?

A

4

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9
Q

What is deltaG? What are its limits?

A

-10 to +10. Indicates the probability of primer secondary structures forming

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10
Q

What are the three steps of PCR?

A

Denaturation, annealing, elongation

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11
Q

What does PCR stand for?

A

Polymerase chain reaction

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12
Q

What occurs during Denaturation? What is its ideal temperature?

A

94 or 95C. H bonds between complimentary bases are broken. Double to single stranded

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13
Q

What occurs during annealing? What is the ideal temperature?

A

Primers bind to their complimentary sequences. Occurs at 55C (52-64avg)or 2-3C lower than Tm

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14
Q

What is another term for annealing?

A

Primer binding

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15
Q

Do the primers become a part of the new PCR product? T/F

A

True!

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16
Q

What are the two types of primers?

A

Forward and reverse complementary

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17
Q

What occurs during the elongation step? At what temperature?

A

DNA polymerase binds to primers and adds dNTPS(new bases) to finish the new strand. Occurs at 72C

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18
Q

What is another term for elongation?

A

Polymerization

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19
Q

How is the elongation step controlled?

A

Time. Length of the step controls the length of complimentary sequence

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20
Q

In what direction does the elongation step occur?

A

5’ to 3’

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21
Q

What is another term for primers?

A

Oligo nucleotides/ oligos

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22
Q

What is the primer directionality? Where does it bind?

A

Bonds to the 3’ end of the template strand and is in the 5’ to 3’ direction

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23
Q

Can forward and reverse primers be different lengths?

A

Yes

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24
Q

Within what range do the annealing temperatures of primers need to be to ensure they both bind?

A

Within 1C

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25
Q

How is the forward primer determined?

A

Right from the start of the sequence, same as the 5’ to 3’ template from the start

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26
Q

How is the reverse primer formed?

A

Compliment of the 5’ to 3’ template on the 3’ end and then “flip it” or find the reverse compliment

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27
Q

Do you compliment the forward primer?

A

NO, only the reverse

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28
Q

What is a restriction enzyme? What does it do?

A

An enzyme that functions in recognition and cutting of DNA sequences

29
Q

Will a restriction enzyme cut DNA if the recognition sequence is changed?

A

No

30
Q

What does the restriction enzyme bind to?

A

Double stranded DNA

31
Q

Where do restriction enzymes cut?

A

Phosphate backbone of DNA

32
Q

How do you determine the concentration of DNA in a sample?

A

Multiply the OD 260 by 50ug DNA/mL

33
Q

What is the ratio that determines a pure DNA sample? What if it is outside of that range?

A

A pure sample is 1.8-2.0. Outside of this range it is contaminated by protein

34
Q

What causes a nonspecific band?

A

When primers bind to the non target region and make smaller copies of different spots in the sequence

35
Q

Is ladder separation linear? T/F

A

True, they move by size

36
Q

How do DNA fragment sizes move in gel?

A

Larger fragments move slower and closer towards black
Smaller fragments move faster and further towards red

37
Q

How long are restriction endonuclease typically?

A

4,5,6,8bp or Bivalent but it is dependent on the target length of the sequence

38
Q

How do you write SNPs?

A

1st possible nucleotide- spot in sequence - 2nd possible nucleotide
C 145 G

39
Q

How many locations differentiate the locations in the gene of a taster vs nontaster?

A

3

40
Q

What does SNP stand for? What does it mean?

A

Single nucleotide polymorphisms, where nucleotides in the sequence differentiate

41
Q

What is Chelex? What if it gets into your PCR sample?

A

Chelex are nano-beads of Mg2+ and Ca2+ that remove metal ions and will inactivate nucleases that will break down DNA. If it gets into your PCR then it inactivates Taq polymerase since its cofactor is Mg

42
Q

What is anthocyanin in plant cross? What gene is this? What expression is dominant?

A

Purple pigment, ANL gene, ANL is dominant over anl

43
Q

What is the ROS gene? What does it indicate? Which expression is dominant?

A

Rosette dwarf gene which gives tall or short plant. Dominant expression is ros/ros

44
Q

What are true breeding parents?

A

Genotypes of homozygous dominant or recessive and we will see the phenotype in all generations. Ex Homozygous dominant ROS/ROS will give tall offspring always

45
Q

What do “fluffy clouds” mean at the bottom of gel electrophoresis runs?

A

Primer dimers

46
Q

What does the percentage of a gel mean in gel electrophoresis?

A

A higher percentage gel means resolving optimal resolution of the linear DNA size you can see

47
Q

What is the purpose of loading dye?

A

To add to DNA samples for PCR to make it sink to the bottom of the well to not affect migration in the buffer and not the gel

48
Q

What is agarose gel composed of?

A

A buffer/salt solution of polymers that sort out DNA fragments by size

49
Q

What is the purpose of agarose gel electrophoresis?

A

To separate DNA fragments by size

50
Q

What is the purpose of the gel solution (2)?

A

Maintains pH and salt concentration

51
Q

Why does DNA move towards the positive (red) electrode (2)?

A

It is negatively charged at a neutral pH

52
Q

What happens when you increase the agarose concentration?

A

Pores become smaller

53
Q

What type of relationship is there between DNA size and migration?

A

Logarithmic relationship between the DNA fragment weights and distance traveled

54
Q

What is the purpose of having dye in the gel itself?

A

To visualize DNA and ladder under UV light

55
Q

What concept do we use when calculating the amount of loading dye?

A

Equivalent volumes

56
Q

Do equivalent volumes equate to 1:1? T/F

A

False!

57
Q

What equation do we use to calculate the amount of loading dye for a reaction?

A

Ve/(n1-n2)
Ve= volume of rxn
N1=initial dye concentration (typically 6x)
N2= final dye concentration (1x)

58
Q

How do we make a cocktail reaction for PCR?

A

Total amount of reactions plus 1 for pipetting error

59
Q

What is typically included in a PCR master mix (4)?

A

dNTPs, Taq, buffer, Mg ions

60
Q

What does “diluted to” mean?

A

The initial volume of solute is diluted to the total volume of solute + diluent
1mL phage is diluted to 4mL =1mL phage +3mL diluent

61
Q

What does “added to” mean?

A

Refers to volume of diluent
1mL phage is added to 4mL broth. 1/5 dilution

62
Q

What is the equation for dilution?

A

D= Vsolute/(Vsolute+Vdiluent)

63
Q

What are the two types of percent solutions?

A

Volume/volume or weight/volume

64
Q

Why do we avoid more than 3 G or C in the last 5bp at the 3’ end of the primer?

A

Avoids primer secondary structure

65
Q

How to go from L to mL? mL to L? L to uL?

A

L to mL *1000
mL to L /1000
L to uL *10^6

66
Q

How to go from M to mM? mM to M? M to uM?

A

M to mM *1000
mM to M /1000
M to uM *10^6

67
Q

What units are used in weight/volume percent solutions?

A

Gr/mL

68
Q

What units are behind molarity?

A

Moles/L

molecular genetics (5 decks)

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