Teknik

Biologi Molekuler
SRI ANGGARINI RASYID
Pokok Bahasan
u Ekstaksi DNA
u Amplifikasi DNA PCR dan
Real-Time PCR
u Elektroforesis
u Design Primer

2
Desain Primer
Mengapa primer penting?

u Primers are what gives PCR/qPCR

its SPECIFICITY!!!
u Good primer design: PCR works great.
u Bad primer design: PCR works terrible.
 Very-Brief PCR Reminder
u PCR is a method to amplify large quantities of a
DNA covering a specific sequence.
 primer
• Primer adalah : oligonukleotida sintetik rantai pendek yang berperan sebagai titik awal sintesa DNA
oleh DNA polymerase. DNA polymerase memulai proses perpanajagan DNA dari ujung 3’
• Target Sequence for PCR Plan to amplify Conventional PCR: 200-800
bp product (~500)

• Real Time PCR: 75-200 bp (~100)

• Short PCR products are typically amplified with higher efficiency than
longer ones; but should be at least 75 bp to easily distinguish from any
primer-dimers
 Good Primer’s Characteristic

Primer length

5..TCAACTTAGCATGATCGGGTAGTAGCTTGACTGTACAACTCAGCAA..3’

18-24 bp for general applications

 General rules for primer design

vPrimer length determines the specificity and significantly affect

its annealing to the template
Ø Too short -- low specificity, resulting in non-specific amplification
Ø Too long -- decrease the template-binding efficiency at normal annealing
temperature due to the higher probability of forming secondary
structures such as hairpins.
Komposisi Basa primer

• Kandungan GC : 50-60% (40-80%)

5 GTGGATGTGGTGTCGATGGC 3

5’ 3’
 Primer melting temperature (Tm):

ü Metling temperature : suhu dimana 50% duplex DNA akan memisah membentuk
untai tunggal
ü Suhu Tm : 52-58 (50-70)
ü TM >65 ada kecenderungan ‘secondary annealing
ü Melting temperature (Tm) : merupakan factor penting untuk menentukan annealing
temperature (Ta)
Wallace rule: Bolton and McCarthy:
Tm = 4 * (G + C) + 2 * (A + T) Tm = 81.5 + 16.6 * Log [I] + 0.41 * (%GC) – 600/L

The nearest neighbor method (Santalucia et.al, 1998):

ΔH (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the
above formula the ΔH is obtained by adding up all the di-nucleotide pairs enthalpy values of each nearest neighbor base pair.
ΔS (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up
all the di-nucleotide pairs entropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor
parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all
calculations.
12
ΔS (salt correction) = ΔS (1M NaCl )+ 0.368 x N x ln([Na+])
 Annealing temperatures

37 – 60oC
gradient
Primer Annealing Temperature

u 3. Primer Annealing Temperature:

Primer Melting temperature : digunakan untuk mengestimasi
stabilitas ikatan DNA-DNA à digunakna untuk menentukan annealing
temperature (Ta)
Ta terlalu tinggi à PCR produk sedikit
Ta terlalu rendah à produk tidak spesifik >>>
u Ta = 0.3 x Tm(primer) + 0.7 Tm (product) – 14.9
u where,

Tm(primer) = Melting Temperature of the primers

Tm(product) = Melting temperature of the product
Stabilitas sekuen pada ujung 3’
• Keberadaan G atau C dalam 5 basa terakhir ujung 3 ‘ (GC clamp) à untuk
meningkatkan annealing ujung 3’

(3’ end should end in at least one G or C (better if there are 2)

More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the
primer.

• Terlalu banyak GC pada ujung 3’ harus dihindari untuk mencegah

misspriming
5 GTGGATGTGGTGTCGATGGC 3 vs CCGATATGCCAGCTATCTGT-3

5’ 5’ 3’
 when is a “primer” a primer?

5’ 3’

5’
3’

5’ 3’

3’
5’
 Primer Pair Matching

• Primers work in pairs – forward primer and reverse primer. Since they are used in
the same PCR reaction, it shall be ensured that the PCR condition is suitable for
both of them.
• One critical feature is their annealing temperatures, which shall be compatible
with each other. The maximum difference allowed is 3 °C. The closer their Tanneal
are, the better.

5 CTGATCAAGTCGATGGCTTG 3 Fw 59 C
5 GATGGAGAGGCTTGACTGC 3 Rv 58 C
 5' CCAGTCGTTACAAACTGAC A 3'
:
::
Avoid Primer Secondary
: || Structures
3' ACAG T CAAACATTGCTGACC 5'

Current- Oligo: no 3'-terminal dimer formation

A. Hairpin It is formed by intramolecular interaction within the
Current+
primer Oligo:
and should the most
be avoided. stable
Optimally dimer
a 3' end hairpinoverall:
with a ΔG of4-2bp, -
kcal/mol
5'and
CCanAinternal hairpin with a ΔG of -33'
G T CGTTACAAACTGACA kcal/mol is tolerated
generally. |||| :
::: :
:::::::
3' ACAG T C A AACATTGCTGACC 5'

Hairpin: ²G = -0.7 kcal/mol, Loop = 8 nt, Tm = 41°

5' CC A G T CGTT
|||| A
3' ACAG T C A AACA
B. Self Dimer:
u A primer self-dimer is formed by intermolecular interactions between the two (same sense)
primers, where the primer is homologous to itself.
u Optimally a 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG
of -6 kcal/mol is tolerated generally.

§ Primer cross dimers are formed by intermolecular interaction between sense and
antisense primers, where they are homologous.
§ Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer
with a ΔG of -6 kcal/mol is tolerated generally
u Repeats: A repeat is a di-nucleotide occurring many times
consecutively and should be avoided because they can
misprime. For example: ATATATAT. A maximum number of di-
nucleotide repeats acceptable in an oligo is 4 di-nucleotides.

u Runs: Primers with long runs of a single base should generally

be avoided as they can misprime. For example,
AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. A
maximum number of runs accepted is 4bp.
Primer cloning

u If you will be including a restriction site at the 5’ end of your

primer, note that a 3-6 base pair "clamp" should be added upstream
in order for the enzyme to cleave efficiently (e.g. GCGGCG-
restriction site-your sequence)
Primer untuk mendeteksi HPV

u Hal yang perlu dipahami sebelum mendesain suatu primer :

u Genom virus
u Perbedaan Genom dalam virion dan saat terintegrasi dalam kromosom
(patogenesa virus)
u Gen yang membedakan antar tipe
u Tujuan à genotyping atau sekuensing whole genome/gen tertentu saja
 Download gene
PaVE: Papilloma virus genome database https://pave.niaid.nih.gov

species group (alpha-9) of the Alphapapillomavirus

Alignmen semua gen
Cari posisi gen yang lestari (conserved)
Cek posisi tersebut di Primer Blast berdasarkan salah satu sekuen L1

Primer Forward
Primer Reverse

5’GATGGTGACATGATGGATATTG3’
Complement 3’CTACCACTGTACTACCTATAAC5’

Reverse Complement 5’CAATATCCATCATGTCACCATC3’

 Forward

Reverse
Hasil analisis Primer Blast
Pembuatan degenerated primer UPAC
nucleotide Base
u Untuk mengakomodasi perbedaan basa antar tipe code
A Adenine
C Cytosine
VVY N G Guanine
Thymine (or
T (or U)
Uracil)
R A or G
Y C or T
S G or C
W A or T
K G or T
M A or C
B C or G or T
D A or G or T
H A or C or T
V A or C or G
AC TAGAAT C C T G T C CAC T GAT G N any base
. or - gap
A C T A G A A T C C T G T V V Y C T N G A T G Degenerated Primers
Thank
You
Jens Martensson

jens@bellowscollege.com