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PBG/MCB 620 DNA Fingerprinting
DNA Fingerprinting
A method for the detection of DNA variation
image source - http://db2.photoresearchers.com/feature/infocus1
Applications of DNA fingerprinting
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Human genetics and disease
Systematics and taxonomy
Population, quantitative, and evolutionary genetics
Plant and animal breeding and genetics
Legal, forensic, and anthropological analysis
Genome mapping and analysis
Important Timeline
•  Discovery of DNA as the Hereditary Material in 1944
•  DNA structure described in 1953
•  Restriction endonucleases discovered in 1968-1969
•  DNA sequencing described in 1977
•  DNA fingerprinting first used in 1985
•  Polymerase chain reaction (PCR) invented in 1985
DeoxyriboNucleic Acid (DNA) structure
“It has not escaped our notice that the specific pairing
we have postulated immediately suggests a possible
copying mechanism for the genetic material”
(Watson and Crick 1953)
DNA Structure
•  DNA is the hereditary material and contains all the information needed to build an
organism.
•  It is a polymeric molecule made from discrete units called nucleotides.
•  Nucleotides link together to form a DNA strand at positions 3’ and 5’
Nitrogenous base:
•  Purines: Adenine and Guanine
•  Pyrimidines: Thymine and Cytosine
Sugar: 2-deoxyribose
Phosphate group
Nucleotide Thymidine
Pairing of Strands
2 strands of polynucleotides:
•  Twisted around each other
in clock-wise direction
•  Antiparallel: complementary
and inverse
•  H-Bridges links that are
specific:
G C
A T
Essential Features of DNA
The structure of DNA is identical in all eukaryotes, therefore the
genetic information resides in the sequence of their bases
Gene is a DNA segment with a sequence of bases that has the
information for a biologic function. Alternative forms of a gene
are called alleles
Location of DNA in Eukaryotic Cells
A small fraction is located in the organelles:
•  Chloroplasts (cpDNA): 135 to 160 kb with high density of genes
•  Mitochondria (mtDNA): 370 to 490 kb. Only about 10% are
genes
Most of it in the nucleus:
Nucleus
•  63 Mb to 150 Gb in plants; 20Mb to 130 Gb in animals
Mitochondria
•  Number of molecules (chromosomes) highly variable: 2 to >500
in animals and 2 to >1000 in plants.
•  Just a very small fraction of the genome is actual genes.
Chloroplast
•  Some tens of thousand genes and gene clusters are scatterd
From Brooker et al. Genetics: Analysis
around in a vast majority of apparently non-functional DNA.
& Principles. McGraw Hill. 2009
•  DNA is associated with other components (mainly proteins) and
form a complex called Chromatin.
DNA Organisation
Chromatin:
The basic structure of chromatin is made of DNA and
proteins (histones)
The structure of the chromatin changes throughout the
cell cycle:
•  Most of the time, when the cell is not undergoing
mitosis, the chromatin is relatively uncondensed.
However, there are more compacted zones
(heterochromatin) and less compacted zones
(euchromatin, which is the majority).
•  When the cell is going to divide, the chromatin gets
more and more compacted producing individualized
structures called methaphasic chromosomes
From Brooker et al. Genetics: Analysis
& Principles. McGraw Hill. 2009
DNA Replication
•  DNA primase: catalyzes the synthesis of a short RNA primer complementary to a single strand
DNA template
•  Helicase: unwinds and separates the two strands of DNA
•  Gyrase: facilitates the action of the helicase relieving tension of the coiled DNA
•  Single Stranded DNA binding proteins (SSB): stabilize single strand DNA
•  DNA polymerase: synthesize a new DNA strand complementary to a template strand by
adding nucleotides one at a time to a 3’ end.
Polymerase Chain Reaction - PCR
•  Invented by K.B Mullis in 1983
•  Allows in vitro amplification of ANY DNA sequence in large numbers
•  Design of two single stranded oligonucleotide primers complementary to
motifs on the template DNA.
PCR Basic Principle
A Polymerase extends the 3’ end of the primer sequence using the DNA strand as
a template.
PCR Principles
•  Each cycle can be repeated multiple times if the 3’ end of the primer is facing the target
amplicon. The reaction is typically repeated 25-50 cycles.
•  Each cycle generates exponential numbers of DNA fragments that are identical copies
of the original DNA strand between the two binding sites.
•  The PCR reaction consists of:
•  A buffer
•  DNA polymerase (thermostable)
•  Deoxyribonucleotide triphosphates (dNTPs)
•  Two primers (oligonucleotides)
•  Template DNA
•  And has the following steps:
•  Denaturing: raising the temperature to 94 C to make DNA single stranded
•  Annealing: lowering the temperature to 35 – 65 C the primers bind to the target
sequences on the template DNA
•  Elongation: DNA polymerase extends the 3’ ends of the primer sequence.
Temperature must be optimal for DNA polymerase activity.
PCR is Exponential
1st
cycle
2nd
cycle
Restriction Endonucleases
•  Enzymes which recognize a specific sequence of bases within double-stranded DNA.
•  Endonucleases make a double-stranded cut at the recognition site.
•  Examples:
EcoRI
HindIII
BamHI
5‘- G|AATTC
5‘- A|AGCTT
5‘- G|GATCC
3‘- CTTAA|G
3‘- TTCGA|A
3‘- CCTAG|G
Gel Electrophoresis
•  A process used to separate
DNA fragments
•  An electric current passes
through agarose or
polyacrylamide gels
•  The electrical current forces
molecules to migrate into the
gel at different rates depending
on their sizes
From Hartwell et al. Genetics. McGraw Hill. 2008
Decoding DNA – Sanger Sequencing
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deoxinucleotyde (dNTP)
Buffer
DNA polymerase
dNTPs
Labeled primer
Target DNA
ddGTP
ddATP
dideoxinucleotyde (ddNTP)
ddCTP
Link:
http://www.wellcome.ac.uk/Education-resources/Education-and-learning/
Resources/Animation/WTDV026689.htm
ddTTP
Reading Sanger Sequencing
*GCTTAAGTACATACCTAGTACCACTATATAATG
G A C T
*GTACATACCTAGTACCACTATATAATG
*GTACCACTATATAATG
*ACGCTTAAGTACATACCTAGTACCACTATATAAT
G
*AAGTACATACCTAGTACCACTATATAATG
*AGTACATACCTAGTACCACTATATAATG
*ATACCTAGTACCACTATATAATG
*ACCTAGTACCACTATATAATG
*AGTACCACTATATAATG
*CGCTTAAGTACATACCTAGTACCACTATATAATG
*CATACCTAGTACCACTATATAATG
*CCTAGTACCACTATATAATG
*CTAGTACCACTATATAATG
*TTAAGTACATACCTAGTACCACTATATAATG
*TAAGTACATACCTAGTACCACTATATAATG
*TACATACCTAGTACCACTATATAATG
*TACCTAGTACCACTATATAATG
*TAGTACCACTATATAATG
Separate gel lanes
Single gel lane
Next Generation Sequencing - Illumina
http://technology.illumina.com/technology/next-generation-sequencing/sequencing-technology.html
Sequencing Options
Method
Read length
Accuracy
Reads per run
Time per run
Cost per 1 million
bases (in US$)
Advantages
Disadvantages
Moderate
throughput.
Equipment can be
very expensive.
5,500 bp to 8,500
Single-molecule
bp avg (10,000 bp);
real-time
maximum read
sequencing (Pacific
length >30,000
Bio)
bases
99.999% consensus
50,000 per SMRT
accuracy; 87%
cell, or ~400
single-read
megabases
accuracy
30 minutes to 2
hours
$0.33–$1.00
Longest read
length. Fast.
Detects 4mC, 5mC,
6mA.
Ion semiconductor
(Ion Torrent
sequencing)
98%
2 hours
$1
Less expensive
equipment. Fast.
Homopolymer
errors.
$10
Long read size.
Fast.
Runs are
expensive.
Homopolymer
errors.
$0.05 to $0.15
Potential for high
sequence yield,
depending upon
sequencer model
and desired
application.
Equipment can be
very expensive.
Requires high
concentrations of
DNA.
$0.13
Low cost per base.
Slower than other
methods. Have
issue sequencing
palindromic
sequence.
$2400
Long individual
reads. Useful for
many applications.
More expensive and
impractical for larger
sequencing
projects.
Pyrosequencing
(454)
up to 400 bp
700 bp
Sequencing by
50 to 300 bp
synthesis (Illumina)
Sequencing by
ligation (SOLiD
sequencing)
Chain termination
(Sanger
sequencing)
99.9%
98%
50+35 or 50+50 bp 99.9%
400 to 900 bp
99.9%
up to 80 million
1 million
24 hours
up to 3 billion
1 to 10 days,
depending upon
sequencer and
specified read
length
1.2 to 1.4 billion
1 to 2 weeks
N/A
20 minutes to 3
hours
Some Plant Sequenced Genomes
Common
name
Year
Chr (#)
Size
Assemble
d
Mb
Assem
Gene (#)
%
Repeat
%
Arabidopsis
2000
5
125
115
92
25,498
14
Rice
2002
12
430
362
84
59,855
26
Sorghum
2009
10
818
739
90
34,496
62
Maize
2009
10
2,300
2048
89
32,540
85
Soybean
2010
20
1,115
973
87
46,430
57
Brachypodium 2010
5
272
272
100
25,532
21
Barley
2012
7
5,100
4980
98
30,400
84
Wheat
2012
21
17,000
3800
22
94,000
80
Barley DNA Sequence
•  Total sequence is 5,300,000,000 base pairs
–  165 % of human genome
–  Enough characters for 11,000 large novels
•  Expressed Genes - 60,000,000 base pairs
–  Approx 1% of total sequence, like humans
–  125 large novels
Finding Sequences
http://www.ncbi.nlm.nih.gov/nuccore
Comparing Sequences
•  Have anonymous from your plant or candidate sequence
from another species
–  What gene does it come from?
•  Compare to existing sequences in databases
–  Basic Local Alignment Search Tool (BLAST)
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Nucleotide query in nucleotide database – BLASTN
Protein query in protein database – BLASTP
Translated nucleotide query in protein database – BLASTX
Protein query in translated nucleotide database – TBLASTN
Translated nucleotide query in translated nucleotide database - TBLASTX
•  Is this consistent with the character?
Multiple Sequence Alignment - Input
www.ebi.ac.uk/Tools/msa/clustalo/
Sequence Alignment - Results
www.ebi.ac.uk/Tools/msa/clustalo/
Gene Expression
DNA SNP/indels genes; molecular markers; epigene.cs
Transcrip.on RNA Transla.on Protein Localisa.on Func.on/Trait Craig Simpson
microarrays
small RNAs; alterna.ve splicing SILAC
Biochemical markers
GFP Localisa.on
Phenotypic traits
Post transcriptional processing
Barley RNA processing linked to abiotic stress responses
•  Evidence that RNA binding proteins and splicing factors respond to abio7c stresses. •  Evidence that abio7c (and bio7c) stress linked genes are alterna7vely spliced. Alterna7ve splicing: •  Increases protein complexity. •  Results in non produc7ve isoforms that counteract transcrip7on. Evidence that most genes which undergo changes in alterna7ve splicing occur independently of transcrip7on. The mechanisms by which environmental condi.ons are translated into post-­‐
transcrip.onal change is essen.al to understanding phenotypic plas.city. Craig Simpson
Alternative Splicing Options
Alternative Splicing
Barley RNA processing linked to abiotic stress responses
Arabidopsis High resolution RT-PCR alternative splicing panel
RT-PCR with ~380 pairs of gene specific primers, one of pair 6-FAM labelled
Separate products by size; quantify the ratio of the products.
Monitor Environmental responses – abiotic stress
F
Standard
Temp.
L
S
Heat Shock
R
At5g41700, Ubiquitin-conjugating enzyme
Craig Simpson
At2g26150 Heat shock responsive expression AS1 AS2