electrophoresis, pcr, dna sequencing, restrictin enzyme

8/8/2019 Electrophoresis, PCR, DNA Sequencing, Restrictin enzyme

1/37

1. Electrophoresis

2. PCR (Polymerase Chain Reaction)

3. DNA Sequencing

4. Restriction Enzyme


2/37

electro : the energy of electricity.

phoresis, from the Greek verbphoros: "to carry across."

1. Gel electrophoresis

Gel electrophoresis is a method that separates macromolecules

either nucleic acids or proteins on the basis of size, electric charge,

and other physical properties, motivated by an electrical current.Separation is done in a gel.

- agarose (DNA and RNA)

- polyacrylamide (DNA, in DNA sequencing; proteins)

often called PAGE (Polyacrylamide Gel Electrophoresis)

2. Free zone electrophoresis

- uses a flow of water, or sometimes a density gradient

collums.

Electrophoresis


3/37

The phosphate groups of DNA and RNA are negatively

charged under most conditions.

T

herefore, DNA and RNA molecules migrate toward a positiveelectrical pole (cathode to anode)

If the electrophoretic medium (a gel) is maintained at a

constant pH, the rate of migration of nucleic acid fragments

toward the positive pole is dependent only on the size of the

fragment, i.e. smaller fragments migrate faster than largerfragments.

Electrophoresis


4/37

Electrophoresis

DNA molecular weight is measured in base pairs (bp), and

commonly in kilobase pairs (kbp; 1,000 bp)

RNA - molecular weight is measured in nucleotide (nt), and

commonly in kilonucleotide (knt)(sometimes bases (b) or kilobases (kb) are used)

Protein - molecular weight is measured in Dalton (Da), and

commonly in kiloDalton (kDa)

The `molecular weight standards` are used to calibrate the

gel run, and the molecular weight of any sample molecule can

be determined by interpolating between the standards.


5/37

Horizontal Electrophoresis

Vertical Electrophoresis


6/37

Applications:Southern Blot

Northern BlotWestern Blot

PCR products

DNA sequencing

RFLP (Restriction Fragment Length Polymorphism)

SSCP (Single Strand Conformational Polymorphism)

Blotting: transferring biological material from a gelonto porous membrane in a way how they were

separated in the gel.

Gel concentration : 1.5%, 1%, 0.8%

Running Buffer:

DNA : TBE (Tris Borate EDTA) / TAE (Tris Acetate EDTA)

RNA : MOPSProtein : SDS

Electrophoresis


7/37

10kb

3kb

0.5kb

DNAmarker

Plasmiduncut

Plasmid(linearized)

Origin(wells)

23kb9.4kb

6.6kb4.5kb

2.3kb

2.0kb

0.56kb

P/HindIII

relaxed

Super-

coiled

RNA

PDNA = 48.36kb

DNA marker= molecular weight standard

Electrophoresis samples


8/37

Staining

DNA Staining DNA is stained with ethidium bromide (EtBr), which

binds to nucleic acids.

The DNA-EtBr complex fluoresces under UV light.

RNA Staining - RNA is also stained with ethidium bromide (EtBr).

Protein Staining Protein is stained with Coomassie Blue (CB).

The protein-CB complex is deep blue and can be

seen with visible light.


9/37

DNA/RNA Blotting

After the DNA, RNA, or protein has been separated by molecular

weight, it must be transferred to a solid support before hybridization.

Usually, the solid support is a sheet of nitrocellulose paper (sometimes

called a filter because the sheet of nitrocellulose were originally used

as filter paper), although other materials are sometimes used.

Southern Blot DNA

Northern Blot RNAWestern Blot - Protein


10/37

OR

A procedure that produces millions of copies of a short

segment of DNA through repeated cycles of:

(Pre-Denaturation)

1) Denaturation

2) Annealing, and

3) Elongation;(Extention)

Polymerase Chain Reaction (PCR):A procedure that is used to amplify a short, well-defined part of a

DNA strand.T

his can be a single gene, or just a part of a gene.


11/37

DNA template: contains the region of the DNA fragment to be amplified.

Forward and Reverse Primer: determine the beginning and end of the region to

be amplified.

dNTP4 : from which the DNA-Polymerase builds the new DNA.Buffer: provides a suitable chemical environment for the DNA-Polymerase.

DNA polymerase: which copies the region to be amplified.

Double strand DNA

(template)

Forward primer Reverse primer

TaqTaq

DNA polymerase

Basic components

of PCR reaction:


12/37

Thermal Cycler / PCR Machines


13/37

Design and order primers PCR machine

PCR MixPCR Cycles PCR tubes

preparation


14/37

Example of PCR cycle

Pre-Denaturation ;90-95oC, 2-5 minutes

Denaturation ; 90-96oC, 1-2 minutes

Annealing; 50-60oC, 1-2 minutes

Elongation, 72oC, 1-2 minutes

Extension, 72oC, 5-10 minutes

Example of PCR mix

DNA template 1 ul

Primers (10 pmoles/ul) 1 ul each

2.5 mM dNTP 4 ul

10x Taq buffer 5 ul

Taq DNA polymerase (1 u/ul) 1 ul

Water to 50 ul

PCR (Polymerase Chain Reaction)


15/37

A. Double

strand DNA

B. Denature96

50

C. Anneal

primers50

D. Polymerase

binds72

Taq

Taq


16/37

72Taq

Taq

E. Copystrands

1

2

3

4

F.

Denature

96

First round

of DNA

synthesis (4

strands)

Taq

Taq


17/37

1

2

3

4

50G. Annealprimers


18/37

1

2

3

4

Taq

Taq

Taq

Taq

72

H.

Polymerase

binds


19/37

1

2

3

4

Taq

Taq

Taq

Taq

I.

Copy

strands

72

Second

round ofDNA

synthesis

(8 strands)


20/37

1

2

3

4

J.

Denature at 96

Anneal primers

at 50


21/37

1

2

3

4

72K.

Bind polymerase (not

shown) and copy

strands

Third

round of

DNA

synthesis(16

strands)


22/37

1

23

4

L.

Denature at 96

Anneal primers

at 50


23/37

1

23

4

M.

Copy strands at

72

Fourth

round of

DNA

synthesis(32

strands)

72


24/37

1

23

4

DNA

strands

(32) are

now

shown as

lines


25/37

1

23

4

After 5 roundsthere are 32

double strands of

which 24 (75%)

are are same

size


26/37

PCR is a very common procedure in molecular genetic and

may be used to:

generate a sufficient quantity of DNA to perform a test

(e.g., DNA sequence analysis)

RAPD (Random Amplified Polymorphic DNA)

AFLP (Amplified Fragment Length Polymorphism)


27/37

Bacterial enzymes that prevent the invasion of foreign DNAs such as

viral DNA, by cutting them up.

These enzymes cut within the foreign DNAs.

These enzymes recognize a specific DNA sequence (4-12bp) which

is twofold symmetry and cut both DNA strands

GAATTC

CTTAAGSome enzymes make staggered cuts

Some make even cuts CCCGGG

GGGCCC

Restriction Endonucleases


28/37

Restriction Endonucleases


29/37

Tohelp protectyour privacy, PowerPointprevented thisexternalpicturefrom being automatically downloaded.To download and display thispicture,click Optionsin the MessageBar, and then click Enableexternalcontent.

Restriction Enzymes and Its Source

Enzyme Source Recognition

Sequence

Cut

EcoRI Escherichia coli 5'GAATTC3'CTTAAG5'---G AATTC---3'3'---CTTAA G---5'

BamHI Bacillus amyloliquefaciens 5'GGATCC3'CCTAGG 5'---G GATCC---3'3'---CCTAG G---5'

HindIII Haemophilus influenzae 5'AAGCTT3'TTCGAA5'---A AGCTT---3'3'---TTCGA A---5'

MstII Microcoleus species 5'CCTNAGG3'GGANTCC

T

aqI Thermus aquaticus

5'TCGA

3'AGCT

5'---T CGA---3

3'---AGC T---5'

NotI Nocardia otitidis 5'GCGGCCGC3'CGCCGGCG

HinfI Haemophilus influenzae 5'GANTC3'CTNAG

AluI* Arthrobacter luteus 5'AGCT3'TCGA5'---AG CT---3'3'---TC GA---5'


30/37

Blund-end Cleavage


31/37

Staggered Cleavage


32/37

Restriction

enzyme

Restriction

enzyme

Ligase

Ligase

DNA ligase covalently links two DNA strands


33/37

Thermal Cycle Sequencing

The DNA to be sequenced is contained

in vector DNA.

Only one primer is used, each with one

dideoxynucleotide (ddA, ddT, ddC, or

ddG) in the reaction mixture. This

generates a series of different chain-

terminated strands, each dependent

on the position of the particular nucleo-tide base where the chain is being

terminated.

After many cycles and with electro-

phoresis, the sequence can be read in

the plate.


34/37

Dideoxy

DNA sequencing


35/37

Automated DNA Sequencing

Involves four fluorophores, one

for each of the four nucleotide

bases.

The resulting fluorescent signal

is recorded at a fixed point when

DNA passes through a capillary

containing an electrophoretic gel.

The sequence is electronically read

and recorded and is visualized as

alternating peaks in one of the four

colors.


36/37

DNA sequencer capillary system

DNA sequencer gel systemNucleotides DNA sequencing

DNA Sequencer


37/37

electrophoresis, pcr, dna sequencing, restrictin enzyme

Documents