3: Bioinformatics PT. 2 Flashcards
→ an extremely powerful technique that enables one to amplify fragments of DNA
PCR
→ an incredibly versatile technique that is applicable to genetic profiling, detection, gene expression, modification, biomedical research, diagnostic testing, and forensic testing
PCR
T or F
successful PCR that produces an optimal amount of product requires the use of good PCR machine
F (requires the use of GOOD PRIMER PAIRS)
The single-most important factor affecting PCR
Choosing appropriate primers
T or F
Primers are important in PCR because it produces specific amplification
T bb quoh
Specific amplification of intended target sequences requires that primers do not have what?
Matches to other targets (allow undesired amplification)
→ 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
Primer
→ aka an oligonucleotide; site where the incoming nucleotides will be added
Primer
cannot start synthesis without a primer
DNA polymerase
T or F
DNA sequence is given as 2 strands
F (given as 1 STRAND ONLY)
T or F
DNA polymerase provides the free 3’-OH group unlike for RNA polymerase
F (Primer provides the free 3’-OH group)
identical to the “top strand” of DNA (5’ to 3’ direction)
forward primer
sequence is in the complementary strand (STILL in 5’ to 3’ direction)
reverse primer
T or F
Reverse primer is derived from the bottom strand which is given by the database
F (NOT GIVEN by the database)
T or F
After getting the reverse primer sequence, that is already enough as a primer sequence
F (NOT ENOUGH kasi you have to turn the sequence around from 3’ –> 5’ to 5’ –> 3’)
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;
target-specific primer
T or F
Target-specific primers only amplifies intended target and is used when DNA sequence is unknown
F (used when DNA sequence is KNOWN)
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
Universal primer
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
16S rRNA gene (rDNA)
T or F
16S rRNA gene (rDNA) is an example of universal primer
T
Familiarize the primers designed for the 16S rRNA gene
16S-F (897-914) - forward primer
16S-FAM-probe (959-977) - negligible;
16S-R (1066-1083) - reverse primer
T or F
16S-FAM-probe (959-977) is still valid sequence used to identify the 16S gene using a probe
F (NEGLIGBLE)
T or F
the different bacteria sequences will be differentiated after PCR
T
T or F
sequences in between the 2 primers does not differ per bacterial species
F (sequences in between the 2 primers GREATLY differ per bacterial species)
→ 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
degenerate primer
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
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)
Familiarize the steps in degenerate primer
- get the DNA sequence by performing PCR/amplification using degenerate primers
- once amplified, get the amplicon and it will reveal the exact sequence of the protein you are working on
Familiarize importance of well-designed primers
- as much an art form as a science
- the success of a PCR reaction relies in part in the specificity of binding of the primer to the template
- a poorly designed primer may amplify DNAs that are not really your targets
WHAT do you do before you start designing a primer?
Check literature (someone may have already designed primers that will do the job for you)
General steps in designing a primer?
- identify amplicon/DNA segment/gene of interest using online databases
- use primer designing software
- check the best primer pair from among the suggested sequences produced from the software
Characteristics of a good primer pair?
- Primer length
- GC content
- GC clamp
Characteristics of a Good Primer Pair:
→ affects both specificity and annealing temperature
→ optimal size: 18-25 nucleotides (aka bases/mers)
primer length
T or F
primer length affects both specificity and annealing temperature
T
Characteristics of a Good Primer Pair: primer length
Optimal size of primers?
18-25 nucleotides
T or F
Bases, mers, and nucleotides all have the same meaning
T
Characteristics of a Good Primer Pair: primer length
Longer primer = ?
shorter primer = ?
Longer primer = unable to anneal to target dna
shorter primer = amplify non-specific regions
T or F
We should avoid more than 3 repeats of bases of the same type to avoid mispriming
T
T or F
AGCGGGGGATGGGG
This sequence is considered long repeats of base which causes mispriming
T
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
GC content
Optimum GC content percentage?
50-60% (can be lower: 40%)
Higher GC content =?
high GC content = higher annealing temperatures
T or F
Higher GC content is a good thing since it produces a good PCR product
F (high gc content has difficulty in annealing/producing a good PCR product because u’re gonna use high temperatures that can denature DNA)
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
GC clamp
Characteristics of a Good Primer Pair: GC Clamp
Presence of G or C bases should be placed within?
within the last 5 bases from the 3’ end of the primers
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
F (within the last 5 bases from the 3’ END of the primers helps promote specific binding at the 3’ END)
Characteristics of a Good Primer Pair: GC Clamp
What should be avoided in GC clamp?
more than 3 G’s or C’s in the last 5 bases at the 3’ end of the primer
Basic rules in primer design?
- melting temperature (Tm)
- complementary sequence
- annealing temperature (Ta)
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
melting temperature (Tm)
T or F
Annealing temperature is the midpoint of temperature range over which the DNA is denatured
F (MELTING TEMP)
Basic rules in primer design:
What is critical in determining annealing temperature?
Melting temperature
T or F
Tm of the 2 primers must be as similar as possible (within 2ºC from each other if possible)
T
Basic rules in primer design: melting temperature
Optimum range of temperature to produce best results?
52-58ºC
Basic rules in primer design: melting temperature
more than 65ºC results in what?
secondary annealing (nonspecific binding of primers)
HOw do you compute for GC content ?
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
HOw do you compute for melting temperature?
Tm (c) = 2 (A + T) + 4 (G + C)
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)
complementary sequence
Basic rules in primer design: complementary sequence
2 types of complementary sequences?
- intraprimer homology
- interprimer homology
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
Intraprimer homology
Basic rules in primer design: 2 types complementary sequence (intraprimer homology)
T or F
presence of double-stranded structures will increase efficiency of annealing
F (presence of double-stranded structures will INTERFERE with the efficiency of annealing)
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
interprimer homology
Basic rules in primer design: 2 types complementary sequence (interprimer homology)
T or F
The primer pair should contain homologous regions/complementary basepairs
F (The primer pair SHOULD NOT contain homologous regions/complementary basepairs as primer dimers may occur)
Basic rules in primer design: 2 types complementary sequence (interprimer homology)
Result if primer pair contains homologous regions?
Primer Dimers
Basic rules in primer design:
→ usually ranges from 50-60ºC
→ should be 5ºC lower than the Tm
annealing temperature (Ta)
Basic rules in primer design: annealing temperature (Ta)
too high annealing temperature = ?
too low annealing temperature = ?
too high annealing temperature = insufficient binding (less amplicons; due to inefficient primer-template hybridization)
too low annealing temperature = non-specific priming
Basic rules in primer design: annealing temperature (Ta)
if non-specific PCR products are obtained, how do you optimize it?
Increasing it stepwise by 1-2ºC both above and below the calculated Ta
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
F (the Ta should be optimized by increasing it stepwise by 1-2ºC both above and below the calculated Ta
Basic rules in primer design: annealing temperature (Ta)
if the calculated Ta is 55ºC, what are the optimized temperatures?
53, 54, 55, 56, and 57ºC (run them separately)
→ 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
Gradient PCR for Optimizing Ta
if u see this card, twerk ka muna
eme pls go see yung visuals ni mam dun sa gradient PCR, yung may bands chechebureche
Familiarize the primers that are sent to companies that synthesize primers
- Integrated DNA Technologies
- Macrogen
- Biorad
- Life Technologies
- Local distributors which may order the primers for you
How to label your primer?
Put a name that will recognize and differentiate between the forward and reverse primers
If your primer is:
Hgb-beta
what should be the label of the F and R primers?
FORWARD: Hgb-beta-F
REVERSE: Hgb-beta-R
T or F
Primers come in a lyophilized (dried powder form) form
T
Steps in reconstituting primers and preparing a working stock?
- centrifuge the tubes before removing the lid to ensure the pellet is at the bottom of the vial
- reconstitute (thaw and dissolve), follow the manufacturer’s instructions
How to prepare master stock?
- add a designated amount of sterile molecular grade water (or autoclaved distilled water) according to how much µL is instructed
- store at -20ºC in a freezer
- 100X (µM)
After preparing master stock, what should be done after?
aliquot the master stock into working stocks: 10X (µM)—diluted 10 times/10 times less concentrated than the working stock
How many times is the master stock diluted to use as working stock
diluted 10 times/10 times LESS concentrated than the working stock
T or F
primers must be optimized to make sure that they are working and specific for your target gene
T
What are the 3 parameters to be optimized for each primer set?
- Annealing temperature (Ta)
- Mg+ concentration - a cofactor of Taq polymerase
- Number of cycles - from 25 to 40 cycles
3 parameters to be optimized for each primer set:
a cofactor of Taq polymerase
Mg+ concentration
3 parameters to be optimized for each primer set:
How many number of cycles is usually needed (range)
from 25 to 40 cycles
