DNA Extraction,

Polymerase Chain
Reaction (PCR) &
Electrophoresis
Yuwono
FAMUS Palembang
 Learning Objectives

Student is able to:

Describe DNA extraction and purification
Describe PCR of a gene
Describe electrophoresis of DNA amplicon
What are the Structures of the Cell?
 EXPERIMENTAL STRATEGIES
Chromosome 11

Blood Samples

DNA extraction

PCR amplification of the gene of interest

Analysis

Results
 Materials
Buffer A
NaAc, pH 5.2 50mM
NaCl 100mM
EDTA 1mM
Add ddH2O

TE
10 mM Tris HCl
1 mM EDTA pH 8.0 (+20C)

PBS pH 7.4
0.5% Saponin in PBS
20% Chelex in ddH2O pH 10.5
 DNA Extraction from Blood Samples
Resuspend final pellet in 4 packed cell volume (PCV) of buffer A
Add 1 PCV of 18% SDS (final concentration of 3%)
Invert to mix and lyse
Leave at room temperature for 2 minutes
Add 6 PCV of phenol/chloroform (25 phenol:24 chloroform:1 iso amyl
alcohol)
Invert to mix
Spin at 2800 rpm for 10 minutes
Keep the aqueous upper phase
Precipitate using 1/10 volume 3M NaAc, pH 5.2 and 2.5 volume
EtOH, overnight at -20C
Spool DNA off with a fired glass pipette and resuspend into 400ul TE
Re-phenol chloroform. Take supernatant into a fresh tube and
precipitate with 1/10 volume 3M NaAc and 2.5 volumes EtOH. Invert
tube to mix. DNA should precipitate. Again spool it into a fresh tube.
Gently resuspend into 500ul TE
 DNA Extraction

Blood Samples

Lysed with 0,5% saponin (10 on ice)

Wash with PBS (3X)

Add Chelex and ddH2O

Boil for 10 minutes

Spin at high speed for 10 mins

DNA containing supernatant

 DNA Extraction from Blood Dotted Paper
Cut about 1 cm2 (divide to 2 or 3 pieces) and put into a sterile1.5 mL
eppendorf tube.
Soak with adding 1 mL of 0.5 % saponin in PBS pH 7.4, then incubate at 4
degrees at least for 4 hours.
Invert the tube for several times.
Spin in high speed for 5 seconds and aspirate the supernatant.
Soak with adding PBS pH 7.4 , then incubate at 4 degrees for at least 20
minutes.
Invert the tube for several times.
Spin at high speed for 5 seconds and aspirate the supernatant.
Push the paper down the tube with a tip.(Dont press it excessively).
Add 50 uL of 20% Chelex and 100 uL of ddH2O
Incubate in boiling water for 10 minutes. Vortex at high speed in each 2-3
minutes during incubation.
Spin at 12 krpm for 5 minutes and collect the DNA containing water into a
new sterile eppendorf tube. (Not worrying if the chelex is carried over).
Spin at 12 krpm for 10 minutes and collect the DNA containing water into
a new sterile tube and use as PCR template or store at -20 degrees. (Avoid
the chelex when collecting DNA).
DNA quality: Clean DNA has a OD260/OD280 between 1.8 and 2.0.

Congratulations!
You have just created your
very own DNA Necklace!
 POLYMERASE CHAIN REACTION (PCR)

A 'licence' to do molecular biology

A key central technique that has revolutionised molecular and
consequently cell biology

devised by Kary Mullis c1983

http://www.nobel.se/chemistry/laureates/1993/mullis-autobio.html
Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction.
Scientific American. 262 (4) 56-65.
WHAT IS PCR?

A simple rapid, sensitive and versatile in vitro method for

selectively amplifying defined sequences/regions of DNA/RNA
from an initial complex source of nucleic acid - generates
sufficient for subsequent analysis and/or manipulation
Human diploid cell contains 6 X 10-9 base pairs
'average' gene size ~ 10,000bp = 1/300,000
600bp fragment = 1/1,000,000
Amplification in a normal PCR will be perhaps a million fold
 APPLICATIONS OF PCR

Cloning of genes or gene fragments

same species or homologous genes from different species (DOP-
PCR)
Genetic diagnosis - Mutation detection
basis for many techniques to detect gene mutations (sequencing)
- 1/6 X 10-9 bp
Paternity testing
Mutagenesis to investigate protein function
Quantitate differences in gene expression
Reverse transcription (RT)-PCR
Identify changes in expression of unknown genes
Differential display (DD)-PCR
Forensic analysis at scene of crime
Industrial quality control
HOW DOES PCR WORK?

Requires the binding of short sequences of DNA

(oligonucleotides/amplimers/primers) to
complementary sequences flanking the desired
target region
the action of an enzyme (DNA polymerase) to
synthesise new exact copies of the target DNA.

5' 3'

3' 5'
5' 3'

3' 5'
DENATURATION
93C - 95C
ANNEALING

37C - 65C

25-35 CYCLES

DENATURATION EXTENSION
93C - 95C
72C
EACH PCR CYCLE HAS THREE STEPS

1. Denaturation; 93C - 95C

30 secs 1min

2. Annealing; 37C - 65C

30 secs 1min
depends on the melting temperature of duplex

3. Extension/Polymerisation; 72C
1min (+ 30secs per 500bp DNA)
TYPICAL REACTION MIXTURE
25 or 50ls in a micro Eppendorf (0.5ml) tube
COMPONENT VOLUME Final
Concentration
10 X PCR Buffer 5l 1X

10 X dNTPs (2mM) 5l 200M

Forward primer (10pmols/l) 5l 1M (50pmols/50l)

Reverse primer (10pmols/l) 5l 1M (50pmols/50l)

Genomic DNA template 2l 1g

Thermostable polymerase 0.5l 1 unit

(2U/l)

H2O (to 50l Final volume) 27.5l

 CYCLING PARAMETERS

Denaturation; 93C - 95C

30 secs 1min
Annealing; 37C - 65C
30 secs 1min depends on the duplex
Extension; 72C
1min
(+ 30secs per 500bp DNA)
25-35 cycles
Final extension 2-10mins
 PCR Agarose gel electrophoresis

3-4 hours

The final product UV visualisation

ALWAYS REMEMBER!
PCR is a highly sensitive technique contamination
with unwanted DNA can be a problem

Always run NEGATIVE controls

Include a positive control if appropriate

Use dedicated filtered tips and positive displacement
pipettes
Dedicated areas?
Can use UV cabinets
 Amplification Exon 11 AE1 Gene

PCR Condition:

PCR Buffer 2.50 mL

MgCl2 0.75 mL
dNTPs 0.50 mL
OVF 1098 0.50 mL
OVR 11272 0.50 mL
ddH2O 18.125 mL
Taq Polymerase 0.125 mL
DNA Template 2.0 mL

Total Volume 25.0 mL

 Amplification Exon 11 AE1 Gene

PCR Reaction
94C 3 minutes
94C 30 second
70C 30 second
70C 3 minutes
30 cycles
 Amplification Exon 11 AE1 Gene
OVF 1098
OVRI 1272

5 ACATCACAGAT GCATTCAGCCCCCAGGTCCTGGCTGCCGTCATCTTCAT
5 ACATCACAGAT . . . . . . . . . . . . . . . . . . . . . . . . . . . GTCATCTTCAT
1197 1225

X/HaeIII 1 2 3

1353 Legends:
X/HaeIII DNA marker
603 Line 1 and 2 Deletion of 27 bp (SAO)
Line 3 No deletion 27 bp
310
281

175
148
DNA Sequencing
 Principles of DNA Sequencing
Primer
DNA fragment

Amp

PBR322

Tet

Ori Denature with Klenow + ddNTP

heat to produce + dNTP + primers
ssDNA
The Secret to Sanger Sequencing
 Principles of DNA Sequencing
5 G C A T G C 3 Template

5 Primer

dATP dATP dATP dATP

dCTP dCTP dCTP dCTP
dGTP dGTP dGTP dGTP
dTTP dTTP dTTP dTTP
ddCTP ddATP ddTTP ddGTP

GddC GCddA GCAddT ddG

GCATGddC GCATddG
 Principles of DNA Sequencing
G T

_ _ short
C A
G
C
A
T
G
C
+ +
long
 Capillary Electrophoresis

Separation by Electro-osmotic Flow

Multiplexed CE with
Fluorescent detection

ABI 3700 96x700 bases

High Throughput DNA
Sequencing
 Large Scale Sequencing
Goal is to determine the nucleic acid
sequence of molecules ranging in size from
a few hundred bp to >109 bp
The methodology requires an extensive
computational analysis of raw data to yield
the final sequence result
 Shotgun Sequencing
High throughput sequencing method that employs
automated sequencing of random DNA fragments
Automated DNA sequencing yields sequences of
500 to 1000 bp in length
To determine longer sequences you obtain
fragmentary sequences and then join them
together by overlapping
Overlapping is an alignment problem, but different
from those we have discussed up to now
 Shotgun Sequencing

Isolate ShearDNA Clone into

Chromosome into Fragments Seq. Vectors Sequence
 Shotgun Sequencing

Sequence Send to Computer Assembled

Chromatogram Sequence
 Analogy
You have 10 copies of a movie
The film has been cut into short pieces with
about 240 frames per piece (10 seconds of
film), at random
Reconstruct the film
 Multi-alignment & Contig
Assembly

ATCGATGCGTAGCAGACTACCGTTACGATGCCTT
TAGCTACGCATCGTCTGATGGCAATGCTACGGAA..

TAGC
TAGCTACGCATCGT C AGAC
AG TACC
GT GTT
TGC
C GA GTTACGATGCCTT
AT
Multiple Sequence Alignment

Multiple alignment of Calcitonins

 Multiple Sequence Alignment
A general method to align and compare
more than 2 sequences
Typically done as a hierarchical
clustering/alignment process where you
match the two most similar sequences and
then use the combined consensus sequence
to identify the next closest sequence with
which to align
 Multiple Alignment Algorithm
Take all n sequences and perform all possible
pairwise (n/2(n-1)) alignments
Identify highest scoring pair, perform an
alignment & create a consensus sequence
Select next most similar sequence and align it to
the initial consensus, regenerate a second
consensus
Repeat step 3 until finished
 Multiple Sequence Alignment
Developed and refined by many (Doolittle,
Barton, Corpet) through the 1980s
Used extensively for extracting hidden
phylogenetic relationships and identifying
sequence families
Powerful tool for extracting new sequence motifs
and signature sequences
Also applicable to DNA contig assembly
 Contig Assembly = Multiple
Alignment
1. Only accept a very high sequence identity
2. Accept unlimited number of end gaps
3. Very high cost for opening internal gaps
4. A short match with high score/residue is
preferred over a long match with low
score/residue
 Contig Assembly Algorithm
Read, edit & trim DNA chromatograms
Remove overlaps & ambiguous calls
Read in all sequence files (10-10,000)
Reverse complement all sequences (doubles # of
sequences to align)
Remove vector sequences (vector trim)
Remove regions of low complexity
Perform multiple sequence alignment
 Contig Alignment - Process
ATCGATGCGTAGC
TAGCAGACTACCGTT
GTTACGATGCCTT
TGCTACGCATCG CGATGCGTAGCA

CGATGCGTAGCA
ATCGATGCGTAGC
TAGCAGACTACCGTT
GTTACGATGCCTT

ATCGATGCGTAGCAGACTACCGTTACGATGCCTT
Reading DNA Chromatograms

Gel ABI Chromatogram

Typical Raw Data
MoKaSih BanYak