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Molecular Biology Lecture > 3: Bioinformatics PT. 2 > Flashcards

3: Bioinformatics PT. 2 Flashcards

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1
Q

→ an extremely powerful technique that enables one to amplify fragments of DNA

A

PCR

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2
Q

→ an incredibly versatile technique that is applicable to genetic profiling, detection, gene expression, modification, biomedical research, diagnostic testing, and forensic testing

A

PCR

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3
Q

T or F

successful PCR that produces an optimal amount of product requires the use of good PCR machine

A

F (requires the use of GOOD PRIMER PAIRS)

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4
Q

The single-most important factor affecting PCR

A

Choosing appropriate primers

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5
Q

T or F

Primers are important in PCR because it produces specific amplification

A

T bb quoh

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6
Q

Specific amplification of intended target sequences requires that primers do not have what?

A

Matches to other targets (allow undesired amplification)

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7
Q

→ short nucleotide sequence that is paired with one strand of DNA and provides a free 3’-OH end at which the polymerase starts synthesis of a DNA chain

A

Primer

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8
Q

→ aka an oligonucleotide; site where the incoming nucleotides will be added

A

Primer

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9
Q

cannot start synthesis without a primer

A

DNA polymerase

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10
Q

T or F

DNA sequence is given as 2 strands

A

F (given as 1 STRAND ONLY)

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10
Q

T or F

DNA polymerase provides the free 3’-OH group unlike for RNA polymerase

A

F (Primer provides the free 3’-OH group)

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11
Q

identical to the “top strand” of DNA (5’ to 3’ direction)

A

forward primer

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12
Q

sequence is in the complementary strand (STILL in 5’ to 3’ direction)

A

reverse primer

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13
Q

T or F

Reverse primer is derived from the bottom strand which is given by the database

A

F (NOT GIVEN by the database)

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14
Q

T or F

After getting the reverse primer sequence, that is already enough as a primer sequence

A

F (NOT ENOUGH kasi you have to turn the sequence around from 3’ –> 5’ to 5’ –> 3’)

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15
Q

Types of Primers:

→ can only anneal to the templates from one species

→ used when the DNA sequence is known (primers in the previous examples)

→ amplifies only the intended target

→ do not have matches to other targets;

A

target-specific primer

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16
Q

T or F

Target-specific primers only amplifies intended target and is used when DNA sequence is unknown

A

F (used when DNA sequence is KNOWN)

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17
Q

Types of Primers:

→ a single sequence that amplifies similar genes related to a specific genus

→ uses only one set of primers but can amplify different types of DNA targets

A

Universal primer

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18
Q

Types of Primers: universalprimer

→ is highly conserved across different species and has a very low evolution rate

→ primers will anneal universally to this

→ assists with differentiating between closely related bacterial species

A

16S rRNA gene (rDNA)

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19
Q

T or F

16S rRNA gene (rDNA) is an example of universal primer

A

T

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20
Q

Familiarize the primers designed for the 16S rRNA gene

A

16S-F (897-914) - forward primer

16S-FAM-probe (959-977) - negligible;

16S-R (1066-1083) - reverse primer

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21
Q

T or F

16S-FAM-probe (959-977) is still valid sequence used to identify the 16S gene using a probe

A

F (NEGLIGBLE)

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22
Q

T or F

the different bacteria sequences will be differentiated after PCR

A

T

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23
Q

T or F

sequences in between the 2 primers does not differ per bacterial species

A

F (sequences in between the 2 primers GREATLY differ per bacterial species)

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24
Q

→ mix or series of primers in which some positions have several possible bases

→ are usually used to amplify DNA fragments based on the available protein sequence

A

degenerate primer

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25
Q

T or F

A codon that are degenerate means that an amino acid may be specified by 1 codon only code for the same amino acid

A

F (amino acid may be specified by MORE THAN 1 CODON OR DIFFERENT CODONS code for the same amino acid OR different codons code for the same amino acid)

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26
Q

Familiarize the steps in degenerate primer

A
  1. get the DNA sequence by performing PCR/amplification using degenerate primers
  2. once amplified, get the amplicon and it will reveal the exact sequence of the protein you are working on
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27
Q

Familiarize importance of well-designed primers

A
  1. as much an art form as a science
  2. the success of a PCR reaction relies in part in the specificity of binding of the primer to the template
  3. a poorly designed primer may amplify DNAs that are not really your targets
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28
Q

WHAT do you do before you start designing a primer?

A

Check literature (someone may have already designed primers that will do the job for you)

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29
Q

General steps in designing a primer?

A
  1. identify amplicon/DNA segment/gene of interest using online databases
  2. use primer designing software
  3. check the best primer pair from among the suggested sequences produced from the software
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30
Q

Characteristics of a good primer pair?

A
  1. Primer length
  2. GC content
  3. GC clamp
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31
Q

Characteristics of a Good Primer Pair:

→ affects both specificity and annealing temperature

→ optimal size: 18-25 nucleotides (aka bases/mers)

A

primer length

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32
Q

T or F

primer length affects both specificity and annealing temperature

A

T

33
Q

Characteristics of a Good Primer Pair: primer length

Optimal size of primers?

A

18-25 nucleotides

34
Q

T or F

Bases, mers, and nucleotides all have the same meaning

A

T

35
Q

Characteristics of a Good Primer Pair: primer length

Longer primer = ?

shorter primer = ?

A

Longer primer = unable to anneal to target dna

shorter primer = amplify non-specific regions

36
Q

T or F

We should avoid more than 3 repeats of bases of the same type to avoid mispriming

A

T

37
Q

T or F

AGCGGGGGATGGGG

This sequence is considered long repeats of base which causes mispriming

A

T

38
Q

Characteristics of a Good Primer Pair:

→ refers to the no. of G’s and C’s in the primer as the percentage of the total bases

A

GC content

39
Q

Optimum GC content percentage?

A

50-60% (can be lower: 40%)

40
Q

Higher GC content =?

A

high GC content = higher annealing temperatures

41
Q

T or F

Higher GC content is a good thing since it produces a good PCR product

A

F (high gc content has difficulty in annealing/producing a good PCR product because u’re gonna use high temperatures that can denature DNA)

42
Q

Characteristics of a Good Primer Pair:

→ the presence of G or C within the last 5 bases from the 3’ end of the primers helps promote specific binding at the 3’ end

→ tighter H-bond between G and C

A

GC clamp

43
Q

Characteristics of a Good Primer Pair: GC Clamp

Presence of G or C bases should be placed within?

A

within the last 5 bases from the 3’ end of the primers

44
Q

T or F

The presence of G or C within the last 5 bases from the 5’ end of the primers helps promote specific binding at the 5’ end

A

F (within the last 5 bases from the 3’ END of the primers helps promote specific binding at the 3’ END)

45
Q

Characteristics of a Good Primer Pair: GC Clamp

What should be avoided in GC clamp?

A

more than 3 G’s or C’s in the last 5 bases at the 3’ end of the primer

46
Q

Basic rules in primer design?

A
  1. melting temperature (Tm)
  2. complementary sequence
  3. annealing temperature (Ta)
47
Q

Basic rules in primer design:

→ the midpoint of temperature range over which the DNA is denatured

→ critical in determining the annealing temperature (Ta)

→ temperature at which one half of the DNA duplex will dissociate to become single stranded

A

melting temperature (Tm)

48
Q

T or F

Annealing temperature is the midpoint of temperature range over which the DNA is denatured

A

F (MELTING TEMP)

49
Q

Basic rules in primer design:

What is critical in determining annealing temperature?

A

Melting temperature

50
Q

T or F

Tm of the 2 primers must be as similar as possible (within 2ºC from each other if possible)

A

T

51
Q

Basic rules in primer design: melting temperature

Optimum range of temperature to produce best results?

A

52-58ºC

52
Q

Basic rules in primer design: melting temperature

more than 65ºC results in what?

A

secondary annealing (nonspecific binding of primers)

53
Q

HOw do you compute for GC content ?

A

derive by counting the G’s and C’s, add them together, and divide them by the total number of bases in sequence; tignan mo nalang formula bb

54
Q

HOw do you compute for melting temperature?

A

Tm (c) = 2 (A + T) + 4 (G + C)

55
Q

Basic rules in primer design:

→ each primer should not have >3 base pairs of intraprimer homology to avoid getting double-stranded structures to form (hairpin formation)

A

complementary sequence

56
Q

Basic rules in primer design: complementary sequence

2 types of complementary sequences?

A
  1. intraprimer homology
  2. interprimer homology
57
Q

Basic rules in primer design: complementary sequence (2 types of complementary sequences)

→ aka self-complementarity - occurs within the same primer

→ there are bases in a single primer that are homologous to the other bases

→ the bases have a tendency to form H-bonds causing hairpin formations

A

Intraprimer homology

58
Q

Basic rules in primer design: 2 types complementary sequence (intraprimer homology)

T or F

presence of double-stranded structures will increase efficiency of annealing

A

F (presence of double-stranded structures will INTERFERE with the efficiency of annealing)

59
Q

Basic rules in primer design: 2 types complementary sequence

→ aka 3’ self complementarity - the 3’ regions of the 2 primers are complementary to each other

→ involves the 2 primers (between the forward and reverse primers)

→ the primer pair should not contain homologous regions (complementary basepairs) as primer dimers may occur

A

interprimer homology

60
Q

Basic rules in primer design: 2 types complementary sequence (interprimer homology)

T or F

The primer pair should contain homologous regions/complementary basepairs

A

F (The primer pair SHOULD NOT contain homologous regions/complementary basepairs as primer dimers may occur)

61
Q

Basic rules in primer design: 2 types complementary sequence (interprimer homology)

Result if primer pair contains homologous regions?

A

Primer Dimers

62
Q

Basic rules in primer design:

→ usually ranges from 50-60ºC

→ should be 5ºC lower than the Tm

A

annealing temperature (Ta)

63
Q

Basic rules in primer design: annealing temperature (Ta)

too high annealing temperature = ?

too low annealing temperature = ?

A

too high annealing temperature = insufficient binding (less amplicons; due to inefficient primer-template hybridization)

too low annealing temperature = non-specific priming

64
Q

Basic rules in primer design: annealing temperature (Ta)

if non-specific PCR products are obtained, how do you optimize it?

A

Increasing it stepwise by 1-2ºC both above and below the calculated Ta

65
Q

Basic rules in primer design: annealing temperature (Ta)

T or F

the Ta should be optimized by increasing it stepwise by 3-4ºC both above and below the calculated Ta

A

F (the Ta should be optimized by increasing it stepwise by 1-2ºC both above and below the calculated Ta

66
Q

Basic rules in primer design: annealing temperature (Ta)

if the calculated Ta is 55ºC, what are the optimized temperatures?

A

53, 54, 55, 56, and 57ºC (run them separately)

67
Q

→ contains different temperature gradients; each pair of the column contains a different temperature zone—helps in testing all possible mixtures at one time

→ the heating blocks have 6 different temperatures that allow for testing of the samples at one time

A

Gradient PCR for Optimizing Ta

68
Q

if u see this card, twerk ka muna

A

eme pls go see yung visuals ni mam dun sa gradient PCR, yung may bands chechebureche

69
Q

Familiarize the primers that are sent to companies that synthesize primers

A
  • Integrated DNA Technologies
  • Macrogen
  • Biorad
  • Life Technologies
  • Local distributors which may order the primers for you
70
Q

How to label your primer?

A

Put a name that will recognize and differentiate between the forward and reverse primers

71
Q

If your primer is:

Hgb-beta

what should be the label of the F and R primers?

A

FORWARD: Hgb-beta-F
REVERSE: Hgb-beta-R

72
Q

T or F

Primers come in a lyophilized (dried powder form) form

A

T

73
Q

Steps in reconstituting primers and preparing a working stock?

A
  1. centrifuge the tubes before removing the lid to ensure the pellet is at the bottom of the vial
  2. reconstitute (thaw and dissolve), follow the manufacturer’s instructions
74
Q

How to prepare master stock?

A
  1. add a designated amount of sterile molecular grade water (or autoclaved distilled water) according to how much µL is instructed
  2. store at -20ºC in a freezer
  3. 100X (µM)
75
Q

After preparing master stock, what should be done after?

A

aliquot the master stock into working stocks: 10X (µM)—diluted 10 times/10 times less concentrated than the working stock

76
Q

How many times is the master stock diluted to use as working stock

A

diluted 10 times/10 times LESS concentrated than the working stock

77
Q

T or F

primers must be optimized to make sure that they are working and specific for your target gene

A

T

78
Q

What are the 3 parameters to be optimized for each primer set?

A
  1. Annealing temperature (Ta)
  2. Mg+ concentration - a cofactor of Taq polymerase
  3. Number of cycles - from 25 to 40 cycles
79
Q

3 parameters to be optimized for each primer set:

a cofactor of Taq polymerase

A

Mg+ concentration

80
Q

3 parameters to be optimized for each primer set:

How many number of cycles is usually needed (range)

A

from 25 to 40 cycles

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