Pcr ppt

Polymerase chain reaction introduction
Material and methods with detailed information .
Application of PCR in different fields.
Disadvantages of traditional PCR in simple way.
Video link for better understanding the PCR method.

1. P r e s e n t e d b y - K u m a r i Jyot i M s c i n b i o t e chnology a n d diploma i n f o r e n s ic science V i n o b a b h a v e u n i v e rsity Topics - P C R ( p o l y m e r a s e c h a i n reaction )

2. Polymerase chain reaction W h a t i s PCR ? Material s a n d m e t h o d A p p l ications of PCR I m p o r t ant formula's u s e d i n PC R T r a d itional p c r d i s advantage . I m p o rtant o f d i f f erent types o f PCR

3. PCR ( polymer a s e chain reaction ) PCR i s in techniques u s e d to m a k e many copies o f s m a l l s e c t i o n o f D N A o r g e n e . I t i s v e r y rapid method f o r a m p l i f i c ation o f desired s e g m ent o f DNA o r RNA . U s i n g p c r i t i s p o s s ible t o g e n e r a t e t h o u s a nds t o m i l l i o n s o f c o p i e s o f p a r t i cular s e g m ent .

4. G e n e rally PCR works i n t h r e e main steps - Denaturation A n n e a l i ng Extinction

5. T e m p e rature r e q u i r e d i n these step's a r e d i f f e r e nt a n d i m p o rtant t o performe PCR .

6. PCR i n s t r u ment

7. T a q p o l ymerase e n z yme a d d t h e n t d e s .

8. M a t e r ial u s e d i n p o l y m erase c h a i n r e a c tion a r e -

9. T a q p o l y m r a s e S o u r c e - T h e r m u s a q u a t i c u s A c t i v i t y - 5 ' - 3 ' polymerase a c t i v i t y ( l a c k s 3 ' - 5 ' e x o n u c l e a s e a c t i v i t i y ) . S t a b i l i t y - h a l f l i f e < 5 m i n a t 1 ° C b u t r e t a i n a c t i v i ty u p t o 4 m i n a t 9 5 ° C . F u n c t i o n - T h e enzyme r e s p o nsible for performing t h e amplification

10. Disadvantage o f T a q p o l ymerase - E r r o r r a t e - 2 × 1 - 4 errors / b a s e I t a l s o l a c k s t h e proofreading a c t i v i t y T a q polymerase i s u n s t able a b o v e 9 ° C .

11. T h e DNA t e m p l a t e i s s t a r t i ng material . I t c o n t ains the r e g ion w h i c h i s amplified . G e n o m i c DNA - 5 - 5 n g i s s u f f icient . P l a s m i d DNA t e m p l a t e - . 1 t o 1 n g i s s u f f i c ient a m o u n t . D N A Template

12. B u f f e r B u f f er p r o v i de a s u i t able e n v ironment f o r DNA p o l e n z y me . T h e pH o f b u f f er i . e 8 . t o 9 . 5 s t a b i l i z e d b y t r i s - H C l . M g C l 2 F u n c t ion a s a cofactor for a c t i v i t y o f DNA p o l ymerase e n z yme . M g 2 + f a c i l i tate s f o r m a t i o n o f t h e c o m p l ex b e t w e e n t h e p r i m e r a n d DNA template b y s t a b i l i z i n g n e g a t ive c h a r g e o n t h e i r phosphate b a c k b o n e .

13. P r i m e r s P r i m e r s a r e short pieces u s u a l l y 2 - 2 5 b p i n l e n g t h . T m - 5 5 t o 7 ° C . 4 t o 6 ℅ G C ( u n i f o r m distribution ) . O n e C a n d G a t 3 ' e n d . I t i s m a d e a s a p a i r - r e v e r s e a n d forward primer . W h i c h a r e b i n d t o the c o m p l e m e n t a r y . s e q u ence .

14. d N T P ' S d N T P s c o n s i s ts - d A T P , d C T P , d G T P , d T T P ( f o u r basic nucleotid es ) . T h e s e a l l a r e added t o p c r r e a ction i n e q u i m o l a r a m o u nt f o r o p t i m a l b a s e i n c o r p o r a t i o n . . 2 m M i s g e n r e a lly u s e d a s f i n a l c o n c e n t r a t i o n . d N T p s exceeding o p t i m a l c o n c entration p r o m o t e m i s i n c o r p o r a t i o n b y n o n - p r o o f r e a d i n g D N A p o l y merase enzyme .

15. Sterile d i s t i l l ed w a t e r P c r g r a d e w a t e r i s p u r e w a t e r . I t i s f r e e f r o m D N a s e a n d R N a s e e n z y m e s . D i s t i l led w a t e r i s used t o n o r m a l i s e t h e t o t a l sample volume .

16. I m p o rtant f o r m u l as - u s e d i n p c r f o r c a l c u l a t i o n o f c o p y n u m b er , and m e l t i n g t e m p erature . C o p y n u m ber = L × n u m b e r o f moles = L × ( t o t a l m a s s / m o l a r m a s s ) . M e l t i ng t e m p e r a t u r e ( T m ) = 2 ( A + T ) + 4 ( G + C )

17. A p p l ications of PCR Molecular i d e n t i f i c a t ion - Genetic m a t c h ing M u t a t i o n s c r e e ning Pathogen detection Molecular A r a c h a e l o g y S e q u e n c ing - B i o i n formatics G e n o m i c c l o n i n g H u m a n g e n o m e project G e n e t ic engineering . G e n e e x p r e s s ions s t u d y F o r e n sic science . P a t e r n i t y testing DNA sample analysis f o r c r i m e scene i n v e s t i g ation .

18. T r a d i t ional p c r d i s a d vantage / l i m i t a t i o n s - E n d p o i n t g e l d e t e c t i o n . P o o r s e n s i tivity S h o r t dynamic r a n g e < 2 f o l d L o w resolution S i z e b a s e d description o n l y . Resu l t s a r e n o t e x p r e s s ed a s n u m b e r E t h i d i u m b r o m i d e f o r s t a i n i n g i s n o t v e r y quali t a tive .

19. Hard t o differentiate b e t w e e n the 1 copies o r 5 c o p i e s o f D N A sample i n g e l

20. G r a p h - i t s h o w t h e phase o f p c r . E x p o n e n t i a l - d o u b l i n g o f p r o d u c t . L i n e a r - t h e r a c t i o n i s s l o w . P l a t e a u - T h e reaction h a d s t o p p e d , n o m o r e p r o d u c t s a r e b e i ng m a d e .

21. N e e d o f different types o f p c r - R e a l t i m e p c r R t - p c r . I n v e r s e p c r Multiplex p c r P c r i n forensic science - A m p F L P . N e s t e d p c r . Primed p c r H o t - s t a r t p c r . I n s i t u p c r . P c r Elisa . ALU p c r

22. https://youtu.be/y3egwMexSTI Video link f o r PCR

23. T h a n k y o u