DNA isolation

1. DNA isolation

2. STEP 1. DNA isolation

4. Polymerase Chain Reaction
(PCR)

5. ‫البلمره‬ ‫تفاعل‬
‫المتسلسل‬
)
Polymerase Chain Reaction (PCR
‫هو‬
‫تقنية‬
‫معملية‬
‫تقوم‬
‫علﻰ‬
‫عمل‬
‫نسﺦ‬
‫عديدة‬
‫من‬
‫ﻗﻄﻌﺔ‬
‫محددة‬
‫م‬
‫ﻦ‬
‫الحمض‬
‫النووي‬
DNA
‫و‬
‫مﻀﺎﻋﻔﺔ‬
‫إنتﺎجه‬
‫ﺣتﻰ‬
‫يمكﻦ‬
‫إجراء‬
‫اﻹ‬
‫ختبﺎرات‬
‫و‬
‫اﻷ‬
‫بحﺎث‬
‫ﻋليه‬
.
‫ﺗمكﻦ‬
‫الﻌﺎلم‬
Dr. Kerry Mullis
‫ﻓﻲ‬
‫ﻋﺎم‬
١٩٨٥
‫م‬
‫مﻦ‬
‫نﺸر‬
‫ﺗقنيﺔ‬
‫الـ‬
PCR
‫ﻓكﺎنﺖ‬
‫هذه‬
‫التقنيﺔ‬
‫بوابﺔ‬
‫لكثير‬
‫مﻦ‬
‫التﻄورات‬
‫المتسﺎرﻋﺔ‬
‫ﻓ‬
‫ﻲ‬
‫مجﺎل‬
‫التكنولوجيﺎ‬
‫الحيويﺔ‬
.

6.  The technique was invented by Dr. Kary Mullis,
1986
 for which he received the Nobel Prize in
Chemistry in 1993.

7. • DNA amplification by PCR (overview)

8. DNA Replication

9. ‫عمل‬ ‫فكرة‬
‫البلمره‬ ‫تفاعل‬
‫المتسلسل‬
)
(PCR
‫درجة‬ ‫إرتفاع‬ ‫يعتمدعلﻰ‬ ‫لتفاعل‬ ‫متعاقبه‬ ‫دوره‬
‫وانخفاضها‬ ‫الحرارة‬
‫لمضاعفة‬
‫من‬ ‫معين‬ ‫جزء‬
‫عدديا‬ ‫الجزء‬ ‫هذا‬ ‫وتضخيم‬ ‫النووي‬ ‫الحمض‬
.

10. PCR Cycle
• Each cycle (Round) of PCR contains 3 steps:
1- Denaturation
2- Primer annealing
3- Primer extension
• The cycle usually repeated for 25 – 40 times.

12. Programming the Thermocycler

13. PCR Melting
94 oC
Temperature
100
0
50
T i m e
5’
3’
3’
5’

14. Melting
94 oC
Temperature
100
0
50
T i m e
3’
5’
5’
3’
Heat

15. PCR Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
3’
5’
5’
3’
5’
5’
Melting
94 oC

16. PCR Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’
5’
5’
3’
Heat
Heat
5’
5’
5’

17. PCR Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’
5’
5’
3’
5’
5’
5’
5’
5’
5’

18. PCR Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’
5’
5’
3’
5’
5’
5’
5’
5’
5’
Heat
Heat

19. PCR Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’
5’
5’
3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

20. Fragments of
defined length
PCR
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’
5’
5’
3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

21. Polymerase Chain Reaction (PCR)
• PCR is a technique which is used to amplify the number of copies of a
specific region of DNA, (usually fewer than 3000 base pairs) in order to
produce enough DNA to be adequately tested.
• Millions of copies of a segment of DNA can be made within a few hours
• As a result, it now becomes possible to analyze and characterize the DNA.

22. THERMOCYCLER
PCR tube
What do we need for PCR?
?

23. ‫البلمره‬ ‫تفاعل‬ ‫تقنيه‬ ‫متطلبات‬
‫المتسلسل‬
)
(PCR
DNA Sample

24. Primers
A primer is a short synthetic oligonucleotide which is used in many
molecular techniques from PCR to DNA sequencing.
These primers are designed to have a sequence which is the reverse
complement of a region of template or target DNA to which we wish
the primer to anneal.

25. Primers
CCGAATGGGATGC
GGCTTACCCTACG
‫نوعان‬
:
•
‫أمامي‬
)
Forward
.(
•
‫ﺧﻠﻔي‬
)
Reverse
.(
‫وهي‬
‫ﺗﺗاﺑﻊ‬
‫واحد‬ ‫شريط‬ ‫في‬ ‫النيﺗروجينيه‬ ‫القواعد‬ ‫من‬
‫قصير‬
)
20-25 b
(
‫في‬ ‫ﺗضﺧيمه‬ ‫المراد‬ ‫الجزء‬ ‫لﺑداية‬ ‫مكمل‬
‫ا‬
‫ل‬
‫ـ‬
DNA
.

27. Taq polymerase
•
‫ﺗسمى‬ ‫ﺑكﺗيريه‬ ‫سﻼلة‬ ‫من‬ ‫مسﺗﺧرج‬
Thermus aquaticus
‫في‬ ‫ﺗعيش‬ ‫الﺗي‬
‫ال‬
‫مياه‬
‫ال‬
‫حارة‬
.
•
‫ﻻ‬
‫يﺗأثر‬
‫ﺑ‬
‫المرﺗﻔعه‬ ‫الحرارة‬ ‫درجات‬
.
•
‫له‬ ‫المثﻠى‬ ‫الحراة‬ ‫درجه‬
٧٢
º
‫م‬
.
First reports using DNA polymerase
from Thermus aquaticus was at (1988)

29. dNTPs

‫أدنين‬
Adenine

‫ثايمين‬
Thymine

‫جوانين‬
Guanine

‫سايﺗوسين‬
Cytosine

30. PCR Buffer 10x
Distilled Water

31. ‫التفاعل‬ ‫نظام‬

33. Making DNA: Components
Cell PCR
ss DNA
template
helicase, etc. ?
dNTPs present present
Primer primase ?
DNA
Polymerase
DNA
polIII
?
Environment nucleus test tube