The document outlines various PCR (Polymerase Chain Reaction) types and their applications in molecular genetics, including colony, nested, multiplex, and long PCR. It also discusses techniques for DNA fingerprinting, such as RAPD and AFLP, as well as reverse transcriptase PCR (RT-PCR) for gene expression analysis and real-time PCR for monitoring reaction progress. Key concepts such as cycle threshold (Ct) values, fluorescence measurement, and melting curve analysis for product verification are also examined.

1. Molecular Genetics
Lecture 4
PCR types

2. • Colony PCR
• Nested PCR
• Multiplex PCR
• Long PCR
• Gradient PCR
• Hot Start PCR
• Inverse PCR
• Overlapping PCR
• DNA fingerprinting
• RAPD
• AFLP
• RFLP
• etc..
• Reverse Transcriptase PCR (RT-PCR)
• Real Time PCR (qRT-PCR)
‫البلمرة‬ ‫لتفاعل‬ ‫الخاصة‬ ‫االنواع‬ ‫بعض‬
‫المتسلسل‬
PCR types

3. salts (ions)
dNTPs
Primers
DNA
Polymerase
Template
DNA
Conventional PCR

4. Colony PCR

5. Nested PCR

6. Multiplex PCR

7. Gradient PCR

8. Hot Start PCR
Used to optimize the yield
of the desired amplified
product in PCR and
simultaneously to suppress
nonspecific amplification.
hotstart
normal

9. Long PCR
Standerd PCR Long PCR
200 bp – 2K bp 5K -20K
Alvin Submersible for
Exploration of Deep Sea
Vents
95ºC 30 S
30-60 ºC 30 S
72 ºC 7min

10. Inverse PCR

‫الـ‬ ‫عزل‬
DNA
‫الخلية‬ ‫من‬ ‫الكلي‬

‫الــ‬ ‫تقطيـع‬
DNA
‫الـي‬ ‫يؤدي‬ ‫الذي‬ ‫القطـع‬ ‫انزيمات‬ ‫احـد‬ ‫بواسـطه‬
‫الــــ‬ ‫مـــن‬ ‫مقاطـــع‬ ‫تكويـــن‬
DNA
‫الزجـــة‬ ‫النهايات‬ ‫نفـــس‬ ‫لهـــا‬
(
Stecky Ends
.)

‫الـ‬ ‫انزيم‬ ‫اضافة‬
ligase
‫مكونة‬ ‫ببعضها‬ ‫النهايات‬ ‫هذه‬ ‫يربط‬ ‫الذي‬
‫الـ‬ ‫من‬ ‫دائرية‬ ‫مقاطع‬
DNA

‫ـة‬‫ق‬‫المنط‬ ‫ـي‬‫ف‬ ‫ـع‬‫ط‬‫بالق‬ ‫يقوم‬ ‫ان‬ ‫ـث‬‫ي‬‫بح‬ ‫ـد‬‫ي‬‫جد‬ ‫ـع‬‫ط‬‫ق‬ ‫ـم‬‫ي‬‫انز‬ ‫ـتخدام‬‫س‬‫ا‬ ‫ـم‬‫ت‬‫ي‬
‫ـي‬‫ل‬‫ا‬ ‫ـه‬‫ق‬‫المعرو‬ ‫ـة‬‫ق‬‫المنط‬ ‫هذه‬ ‫انتقال‬ ‫ـي‬‫ل‬‫ا‬ ‫يؤدي‬ ‫ـا‬‫م‬‫م‬ ‫ـع‬‫ب‬‫التتا‬ ‫ـة‬‫ف‬‫معرو‬
‫المقطع‬ ‫اطراف‬

‫الــ‬ ‫تفاعـل‬ ‫اثناء‬ ‫كقالـب‬ ‫القطـع‬ ‫هذه‬ ‫تسـتخدم‬
PCR
‫ترتبـط‬ ‫بحيـث‬
‫االطراف‬ ‫علي‬ ‫توجد‬ ‫التي‬ ‫التتابع‬ ‫معروفه‬ ‫المنطقه‬ ‫في‬ ‫البرايمرز‬

‫الـ‬ ‫بواســطة‬ ‫المقطــع‬ ‫بهذا‬ ‫الخاص‬ ‫التتابــع‬ ‫تحديــد‬ ‫يتــم‬
DNA
sequencer

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

12. Overlapping PCR
PCR 1 PCR 2
‫الـ‬ ‫ن‬‫م‬ ‫ل‬‫ك‬ ‫ج‬‫نوات‬ ‫ط‬‫خل‬ ‫م‬‫يت‬
PCR1
‫الـ‬ ‫و‬
PCR2
‫م‬ ‫يت‬ ‫و‬ ‫جديدة‬ ‫ه‬ ‫انبوب‬ ‫ي‬ ‫ف‬
‫للـ‬ ‫ب‬ ‫كقال‬ ‫تخدامهما‬ ‫اس‬
DNA
‫الـ‬ ‫ي‬ ‫ف‬
PCR3
PCR 3

13. AFLP
RAPD
S
S
R
ISSR
R
F
L
P
MASAP
VNTR
CAPS
S
R
A
P
SSCP
SNP
COS
DArT
S
C
A
R
RDA
S
A
M
P
L
Molecular Markers
(DNA fingerprinting)

14. Restriction Fragment Length Polymorphism
(RFLP)

15. Restriction Fragment Length Polymorphism
(RFLP)
Restriction site?
Restriction Enzymes: E.g.,
EcoR1

17. Randomly amplified polymorphic DNA
RAPD

18. Amplified Fragment Length Polymorphism
AFLP-PCR

19. Reverse transcription

20. qRT-PCR is used to qualitatively detect gene expression
Why RT-PCR?
 Gene expression analysis
 Cancer research
 Drug research
 Disease diagnosis and management
 Viral quantification
 Food testing
 Percent GMO food
 Animal and plant breeding
 Gene copy number

22. RNA AAAAAAAAAAAA
TTTTTT
oligo dT primer
All messenger RNAs (mRNA) have a poly A tail
The RT reaction can be primed by a:
target specific primer (i.e. primer
targeting VHSV nucleocapsid (N)
gene)
oligo dT primer (a primer consisting
of a run of T’s that targets the mRNA
poly A tail)
random primers (a mix of 6 base
primers consisting of random
nucleotides)

23. RT-PCR
mRNA
oligo dT
RT: Reverse transcriptase
cDNA
mRNA
5´
3´
5´ 3´
Templat for RT- PCR

24.  Real-Time PCR a specialized technique that allows a PCR
reaction to be visualized “in real time” as the reaction
progresses.
 Quantitative PCR relies on the principal that the quantity
of target at the start of the reaction is proportional to
amount of product produced during the exponential
phase
∆
Fluorescence
CT CT
Greater starting target
Less starting target
<
Real-Time PCR

25. How does PCR work?
Log
Target
DNA
Cycle Number
Theoretical

26. Conventional PCR problem
Log
Target
Agarose gel
electrophoresis
following PCR
Cycle Number

27. Initial DNA strand
First PCR cycle
Second PCR cycle
Third PCR cycle
Fourth PCR cycle
How to measure the PCR product?

29. Any increase in fluorescence level can be plotted onto a
graph and easily interpreted
Virus present
No virus
Cycles
Fluorescence
level

30. 0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43
Cycle
Fluorescence
40
50
60
70
80
90
100
Temperature
Denaturation
Extension Quantitate
Melting
curve
Annealing

31. We describe the position of the lines with a value that represents
the cycle number where the trace crosses a threshold.
This is called the cycle threshold “Ct Value”.
Ct values are directly related to the starting quantity of DNA, by
way of the formula:
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
23 25 28
Ct Values:

32. Quantitative PCR – in depth
 Major assay types
 Hydrolysis probes
 Basis of TaqMan® chemistry
 Uses two primers and an internal hydrolysis
probe
 Most commonly used for fish health
diagnostics
 SYBR ® green dye
 Increased fluorescence when bound to dsDNA
 Slightly lower specificity
 Costs less
 May not be as sensitive as the 5’ nuclease
assays

33. SYBRGreen

34. PCR makes more double-
stranded DNA
SYBR Green dye
binds to dsDNA
When illuminated with light at 490nm, the
SYBR+DNA complex fluoresces at 520nm.
5’ 3’
5’
3’
5’ 3’
5’
3’
5’ 3’
5’
3’
5’
5’
Taq
Taq
3’
5’
3’
Taq
Taq
5’
5’
l l l
l
l

36. 40
50
60
70
80
90
100
Temperature
Denaturation
Extension Quantitate
Melting
curve
Annealing

37. 5’ 5’
3’
3’
5’
5’
3’
3’
ID ID
ID
5’
5’
3’
3’ ID
• Melt curves can tell us what products are in a
reaction.
• PCR products that are shorter will melt at lower
temperatures.
• Different PCR products will therefore have different
shaped curves.