PRIMER DESIGN

Question 1

● Single-Stranded DNA Fragments
● 20-30 bp long

Answer 1

PRIMERS

Question 2

TWO TYPES:

Answer 2

1. Forward Primer
2. Reverse Primer

Question 3

● 3’ to the sequences amplified
● Hybridizes with the plus strand

Answer 3

REVERSE PRIMER

Question 4

● 5’ to the sequences amplified
● Hybridizes with the minus strand

Answer 4

FORWARD PRIMER

Question 5

Creation of Short, oligonucleotide sequences to be used in amplifying a specific region of DNA

Answer 5

PRIMER DESIGN

Question 6

WHAT ARE THE FACTORS CONCERNING THE PRIMER

Answer 6

1. Misprimes
2. Primer Dimers

Question 7

● Artifact in PCR
● PCR products double the size of primers
● Primers binding to each other

Answer 7

PRIMER DIMERS

Question 8

● Aberrant binding of the primer
● Carries the primer sequence and becomes a target
for the next amplifications

Answer 8

MISPRIMES

Question 9

DESIGNING THE PRIMER
GIVE ME THE 1ST STEP AND EXPLAIN BRIEFLY

Answer 9

I. IDENTIFY YOUR TARGET
● Search for a pre-existing sequence similar to yours
● Use known sequences for the organism for metagenomics/shotgun sequencing

Question 10

DESIGNING THE PRIMER
GIVE ME THE 2ND STEP AND EXPLAIN BRIEFLY

Answer 10

II. FIND FLANKING REGIONS
● Check the FASTA file of your region of interest
● include several hundred bases upstream and downstream

Question 11

DESIGNING THE PRIMER
GIVE ME THE 3RD STEP AND EXPLAIN BRIEFLY

Answer 11

III. PRIMER BLAST
● Use the FASTA file with the flanking regions for a primer design software
● Check the different parameters

Question 12

DESIGNING THE PRIMER
GIVE ME THE 4TH STEP AND EXPLAIN BRIEFLY

Answer 12

IV. PICK AND ASSESS
● Pick a primer pair or several primer pairs to study further based on criteria: (Criteria below)
● Use a sequence analyzer to predict possible secondary structures or primer-dimers

Question 13

T or F
PRIMER DESIGN GUIDELINES/CRITERIA:
Supplementary to flanking sequences of target

Answer 13

F - Complementary

Question 14

PRIMER DESIGN GUIDELINES/CRITERIA:
Primer length: ___

Answer 14

18-22 bp

Question 15

PRIMER DESIGN GUIDELINES/CRITERIA:
Primer melting temperature.
what is the optimal temp?

Answer 15

52-58 C is optimal

Question 16

PRIMER DESIGN GUIDELINES/CRITERIA:
Estimation: Melting Temperature: =

Answer 16

(G+C)x4 + (A+T)x2
this is in celcius

Question 17

PRIMER DESIGN GUIDELINES/CRITERIA:
this estimates how stable your dna-dna hybrid will be or how well it will stay together after annealing

Answer 17

Annealing temperature

Question 18

PRIMER DESIGN GUIDELINES/CRITERIA:
GC content: __

Answer 18

40-60%

Question 19

PRIMER DESIGN GUIDELINES/CRITERIA:
GC clamp: ___

Answer 19

1-2 G/C at 3’ end

Question 20

T or F
single base runs should be practiced

Answer 20

F - Dinucleotide Repeats or single base runs should be avoided

Question 21

T or F
Avoid cross-homology with other sites of the sequence

Answer 21

T

Question 22

T or F
Avoid primer secondary structures

Answer 22

T

Question 23

T or F
Avoid self complementarity and 5’
complementarity

Answer 23

F - 3’

Question 24

DESIGNING THE PRIMER
GIVE ME THE 5TH STEP AND EXPLAIN BRIEFLY

Answer 24

V. MAKE MORE PRIMERS
● Not needed if the entire sequence is already accounted for
● Sometimes, another primer pair must be made to sequence an entire gene

Question 25

DESIGNING THE PRIMER
GIVE ME THE 6TH STEP AND EXPLAIN BRIEFLY

Answer 25

VI. PUT DOWN YOUR ORDER
● Order your primer sequences and wait for them to
arrive:
○ BioRad
○ Thermofisher
○ Integrated DNA technologies (IDT)
○ Eurofins