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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
• 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
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
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
WHERE IS THE DNA LOCATED IN EUKARYOTES?
A small fraction is located in the organelles:
• Chloroplats (cpDNA): 135 to 160 kb with high density of genes
• Mithocondria (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.
Chloroplast
• Just a very small fraction of the genome is actual genes.
• Some tens of thousand genes and gene clusters are scatterd
From Brooker et al. Genetics: Analysis
& Principles. McGraw Hill. 2009
around in a vast majority of apparently non-functional DNA.
• DNA is associated with other components (mainly proteins) and
form a complex called Chromatin.
WHERE IS THE DNA LOCATED IN
EUKARYOTES?
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 MUTATIONS
Changes in the nucleotide sequence of genomic DNA that
can be transmitted to the descendants.
If these changes occur in the sequence of a gene, it is
called a mutant allele. The most frequent allele is called
the wild type.
A DNA sequence is polymorphic if there is variation
among the individuals of the population.
DNA MUTATIONS
Types of mutations depending on the effect on the DNA sequence:
Wildtype
5’ – AGCTGAACTCGACCTCGCGATCCGTAGTTAGACTAG -3’
Substitution
(transition: A
5’ – AGCTGAACTCGGCCTCGCGATCCGTAGTTAGACTAG -3’
G
Substitution
(transversion: G
5’ – AGCTCAACTCGACCTCGCGATCCGTAGTTAGACTAG -3’
C)
C
Deletion
(single bp)
5’ – AGCTAACTCGACCTCGCGATCCGTAGTTAGACTAG -3’
CAACTCGACC
Deletion
(DNA segment)
5’ – AGCTTCGCGATCCGTAGTTAGACTAG -3’
DNA MUTATIONS
Types of mutations depending on the effect on the DNA sequence:
Wildtype
5’ – AGCTGAACTCGACCTCGCGATCCGTAGTTAGACTAG - 3’
Insertion
(single bp)
5’ – AGCTGAACTACGACCTCGCGATCCGTAGTTAGACTAG - 3’
Insertion
(DNA segment)
5’ – AGCTGAACTAGTCTGCCCGACCTCGCGATCCGTAGTTAGACTAG -3’
Inversion
5’ – AGCAGTTGACGACCTCGCGATCCGTAGTTAGACTAG -3’
Tranposition:
5’ – AGCTCGACCTCGCGATCCGTAGTTATGAACGACTAG - 3’
DNA MUTATIONS
Types of mutations depending on the effect on the protein:
Wildtype
5’ – AGCTCAACTCGACCTCGCGATCCGAAGTTAGACTAG - 3’
Ser
Silent
Thr
Arg
Pro
Arg
Asp
Pro
Lys
Leu
Asp STOP
5’ – AGCTCAACTCGACCTCGTGATCCGAAGTTAGACTAG - 3’
Ser
Amino acid
change
Ser
Ser
Thr
Arg
Pro
Arg
Asp
Pro
Lys
Leu
Asp STOP
5’ – AGCTCAACTCGACCTTGCGATCCGAAGTTAGACTAG - 3’
Ser
Ser
Thr
Arg
Pro
Cys
Asp
Pro
Lys
Leu
Asp STOP
A
Frame shift
5’ – AGCTCAACTCGCCTCGCGATCCGAAGTTAGACTAG - 3’
Ser
STOP
Ser
Thr
Arg
Leu
Ala
Ile
Arg
Ser
STOP
5’ – AGCTCAACTCGACCTCGCGATCCGTAGTTAGACTAG - 3’
Ser
Ser
Thr
Arg
Pro
Arg
Asp
Pro
Lys
REPLICATION
TRANSCRIPTION
DNA
TRANSLATION
RNA
PROTEINS
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.
A Polymerase extends the 3’ end of the primer sequence using the DNA strand as
a template.
POLYMERASE CHAIN REACTION – PCR
• 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)
• Deoxyrybonucleotide triphospates (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.
1st
cycle
2nd
cycle
POLYMERASE CHAIN REACTION – Links:
•
•
http://www.dnalc.org/resources/animations/pcr.html
http://learn.genetics.utah.edu/content/labs/pcr/
3rd
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
• 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
SANGER DNA SEQUENCING
Link: http://www.wellcome.ac.uk/Education-resources/Teaching-andeducation/Animations/DNA/WTDV026689.htm
•
•
•
•
•
deoxinucleotyde (dNTP)
Buffer
DNA polymerase
dNTPs
Labeled primer
Target DNA
ddGTP
ddATP
dideoxinucleotyde (ddNTP)
ddCTP
ddTTP
*GCTTAAGTACATACCTAGTACCACTATATAATG
G A C T
*GTACATACCTAGTACCACTATATAATG
*GTACCACTATATAATG
*ACGCTTAAGTACATACCTAGTACCACTATATAATG
*AAGTACATACCTAGTACCACTATATAATG
*AGTACATACCTAGTACCACTATATAATG
*ATACCTAGTACCACTATATAATG
*ACCTAGTACCACTATATAATG
*AGTACCACTATATAATG
*CGCTTAAGTACATACCTAGTACCACTATATAATG
*CATACCTAGTACCACTATATAATG
*CCTAGTACCACTATATAATG
*CTAGTACCACTATATAATG
*TTAAGTACATACCTAGTACCACTATATAATG
*TAAGTACATACCTAGTACCACTATATAATG
*TACATACCTAGTACCACTATATAATG
*TACCTAGTACCACTATATAATG
*TAGTACCACTATATAATG
Separate gel lanes
Single gel lane
DNA polymorphisms
• Insertion-deletion length polymorphism – INDEL
• Single nucleotide polymorphism – SNP
• Simple sequence repeat length polymorphism –
mini- and micro-satellites
A C A T T GCGAA T T C A T GT A CGC A T
T GT AA CGC T T AAGT A CA T GCGT A
A C A T T GCGAAG T C A T GT A CGC A T
T GT AA CGC T T C AGT A CA T GCGT A
Allele A
Allele a
A
a
a
A
a
a
A
a
Ind 1 Ind 2 Ind 3 Ind 4 Ind 5 Ind 6 Ind 7 Ind 8
A C A T T GCGAA T T C A T GT A CGC A T
T GT AA CGC T T AAGT A CA T GCGT A
A C A T T GCGAAG T C A T GT A CGC A T
T GT AA CGC T T C AGT A CA T GCGT A
Allele A
Allele a
A
a
a
A
a
a
A
a
Ind 1 Ind 2 Ind 3 Ind 4 Ind 5 Ind 6 Ind 7 Ind 8
Restriction Fragment Length
Polymorphism (RFLP)
• RFLPs (Botstein et al. 1980) are differences in restriction
fragment lengths caused by a SNP or INDEL that create
or abolish restriction endonuclease recognition sites.
• RFLP assays are based on hybridization of a labeled DNA
probe to a Southern blot (Southern 1975) of DNA digested
with a restriction endonuclease
Labeled
Probe
Target
3’ TGGCTAGCT 5’
1
3’ TGGCTAGCT 5’
|||||||||
5’-CCTAACCGATCGACTGAC-3’
2
5’-GGATTGGCTAGCTGACTG-3’
RFLP
Features of RFLPs
•
•
•
•
•
•
Co-dominant
Locus-specific
Genes can be mapped directly
Supply of probes and markers is unlimited
Highly reproducible
Requires no special instrumentation