SNPs, Polymorphism, PCR, Electrophoresis, RFLP

1. -SAURABH SHUKLA
&
MAYANK JAIN

2. Things to be learned:
Polymorphism
SNPs
DNA isolation
Polymerase chain reaction (PCR)
Gel electrophoresis
RFLP (or Restriction fragment length polymorphism)

3. Polymorphism

4. Polymorphism
•When alleles are so common that they are found in
more than 1% of chromosomes in the general
population, the alleles constitute what is known as
genetic polymorphism
•while less than 1% constitute mutations
•That includes SNPs, insertions, deletions
•The human genome contains 1.42 million SNPs.

5.  A polymorphism is a clinically harmless DNA variation that
does not affect the phenotype.
 At the molecular level, polymorphism is a variation in
nucleotide sequence from one individual to another. SNP or
SNV
 Polymorphisms often occur in the intervening sequences that
do not code for proteins. [Note: Only a few percent of the
human genome actually encodes proteins.]
 All the common blood types, such as the ABO blood group
system , are genetic polymorphisms.

6. SNPs
•A SNP ("snip") is a single base mutation in DNA.
•SNPs are the most simple form and most common
source of genetic polymorphism in the human
genome (90% of all human DNA polymorphisms).

7. DETECTION
OF
SNPs

8. 1) DNA extraction
2) PCR
3) RFLP

9. DNA Isolation:

10. Lysis:
• Break down cells to access DNA in the
nucleus
• Lysis buffer
• Chaotropic salts
• Destabilise hydrogen bonds,
hydrophobic interactions,
proteins (including
nucleases)
• Disrupts the association of
nucleic acids with water in
preparation to bind to silica
membrane
• Heat

11. Purification – Bind:
• Ethanol is added to enhance the
binding of DNA to silica
• Load sample to column
• Centrifuge
• DNA binds to the membrane and
remaining lysate discarded

12. Purification – Wash:
 There are typically 2 washes with a centrifuge step after
each
 Wash 1 will contain a low amount of chaotropic salt to
remove any remaining proteins and coloured contaminants
 Wash 2 contains a high concentration of ethanol to remove
the remaining salts.
 Salts MUST be removed for good DNA yields and purity.
Wash 2 can be repeated to ensure this.
 All ethanol MUST be removed so that the DNA can be
successfully removed/eluted from the silica membrane.

13. DNA Isolated:

15. Types of PCR
Long PCR:
 Used to amplify PCR products upto 27kb .
Nested PCR:
 Involves two consecutive PCR reactions of 25 cycles.
 The first PCR uses primers external to the sequence of interest.
 The second PCR uses the product of the first PCR

16. RT-PCR (Reverse Transcription PCR) :
•Used to reverse-transcribe and amplify RNA to cDNA.
•PCR is preceded by a reaction using reverse transcriptase, an enzyme
that converts RNA into cDNA.
•The two reactions may be combined in a tube.
•Uses: 1-Detection of RNA virus like (HCV).
Quantitative Real-Time PCR (qRT-PCR) : Method use fluorescent
dyes, such as Sybr Green, or fluorescence-containing DNA probes, such
as TaqMan, to measure the amount of amplified product as the
amplification progresses

17. PCR Reagents
• Template DNA
• Primers: are synthesized by software like GENETOOL
• dNTPs
• DNA Polymerase
• PCR reaction buffer
• Thermocyler
Finding the TM value of primer – TM = {4(G+C)}+{2(A+T)}
Fig 1 – Amplification of the DNA

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

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

20. Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
3’5’
5’3’
5’
5’
Melting
94 oC

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

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

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

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

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

26. PCR machine
NEW AUTOMATED PCR

27. PCR Products:

28. Restriction Fragment Length Polymorphism
 A restriction fragment length polymorphism (RFLP)
is a genetic variant that can be examined by cleaving
the DNA into fragments (restriction fragments) with a
restriction enzyme.
 An inherited difference due to genomic variations in
the pattern of restriction enzyme digestion is known
as a RFLP.

29. Genomic variations
 Genome variations are differences in the sequence of
DNA among individuals that do not affect the
phenotype.
 the genomes of nonrelated people differ at about 1 of
1,500 DNA bases, or about 0.1% of the genome.

30. What is:
 A restriction enzyme(s)?
 Type I,II and III
 An endonuclease
 We will focus on type II.
 A restriction digest?

31. Restriction Enzyme:

32. Restriction Enzyme Digest:

33. Restriction Enzyme Digest:
 Place samples at 37C for 1 hour
 After 1 hour you will be ready to load
your gel

34. Example of known sizes of DNA
DNA Ladder Markers

35. Gel Electrophoresis:
 Load max. 25 ul per well
 Run gel at 75 volts until the dye front is approximately
half-way down gel.
 Take photograph

36. Agarose Electrophoresis Loading
• Electrical current
carries
negatively-
charged DNA
through gel
towards positive
(red) electrode
Power Supply
Buffer
Dyes
Agarose gel

37.  Agarose gel
sieves DNA
fragments
according to size
 Small
fragments move
farther than
large fragments
Power Supply
Gel running
Agarose Electrophoresis Loading

38. Restriction Digest:
 AFTER 1 hour DIGESTION: You must add 3ul 6X loading dye to
your samples (not to the ladder (L)).

39. Analysis of Data:
•Allows you to identify sizes of DNA
•By comparing migration of digested PCR product.

40. How do we get wild type or mutant?

41. Thank you
THANK YOU