DNA Fingerprinting A method for the detection of DNA variation PBG/MCB 620 DNA Fingerprinting image...

DNA Fingerprinting A method for the detection of DNA variation

PBG/MCB 620 DNA Fingerprinting

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

Nitrogenous base:• Purines: Adenine and Guanine• Pyrimidines: Thymine and Cytosine

Sugar: 2-deoxyribose

Phosphate group

• 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’

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:

• 63 Mb to 150 Gb in plants; 20Mb to 130 Gb in animals• 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.• Some tens of thousand genes and gene clusters are scatterd

around in a vast majority of apparently non-functional DNA.• DNA is associated with other components (mainly proteins) and

form a complex called Chromatin.

Nucleus

Chloroplast

Mitochondria

From Brooker et al. Genetics: Analysis & Principles. McGraw Hill. 2009

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:

5’ – AGCTGAACTCGACCTCGCGATCCGTAGTTAGACTAG -3’Wildtype

5’ – AGCTGAACTCGGCCTCGCGATCCGTAGTTAGACTAG -3’Substitution(transition: A G

5’ – AGCTCAACTCGACCTCGCGATCCGTAGTTAGACTAG -3’Substitution(transversion: G C)

5’ – AGCTAACTCGACCTCGCGATCCGTAGTTAGACTAG -3’Deletion(single bp)

C

5’ – AGCTTCGCGATCCGTAGTTAGACTAG -3’Deletion(DNA segment)

CAACTCGACC

DNA MUTATIONS

Types of mutations depending on the effect on the DNA sequence:

5’ – AGCTGAACTCGACCTCGCGATCCGTAGTTAGACTAG - 3’Wildtype

5’ – AGCTGAACTACGACCTCGCGATCCGTAGTTAGACTAG - 3’Insertion(single bp)

5’ – AGCTGAACTAGTCTGCCCGACCTCGCGATCCGTAGTTAGACTAG -3’Insertion(DNA segment)

5’ – AGCAGTTGACGACCTCGCGATCCGTAGTTAGACTAG -3’Inversion

Tranposition: 5’ – AGCTCGACCTCGCGATCCGTAGTTATGAACGACTAG - 3’

DNA MUTATIONS

Types of mutations depending on the effect on the protein:

5’ – AGCTCAACTCGACCTCGCGATCCGAAGTTAGACTAG - 3’WildtypeSer Ser Thr Arg Pro Arg Asp Pro Lys Leu Asp STOP

5’ – AGCTCAACTCGACCTCGTGATCCGAAGTTAGACTAG - 3’SilentSer Ser Thr Arg Pro Arg Asp Pro Lys Leu Asp STOP

5’ – AGCTCAACTCGACCTTGCGATCCGAAGTTAGACTAG - 3’Amino acid change Ser Ser Thr Arg Pro Cys Asp Pro Lys Leu Asp STOP

5’ – AGCTCAACTCGCCTCGCGATCCGAAGTTAGACTAG - 3’Frame shiftSer Ser Thr Arg Leu Ala Ile Arg Ser STOP

5’ – AGCTCAACTCGACCTCGCGATCCGTAGTTAGACTAG - 3’STOPSer Ser Thr Arg Pro Arg Asp Pro Lys

A

DNA RNA PROTEINS

REPLICATION

TRANSCRIPTION TRANSLATION

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

5‘- G|AATTC

3‘- CTTAA|G

HindIII

5‘- A|AGCTT

3‘- TTCGA|A

BamHI

5‘- G|GATCC

3‘- CCTAG|G

From Hartwell et al. Genetics. McGraw Hill. 2008

• 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

SANGER DNA SEQUENCING

deoxinucleotyde (dNTP) dideoxinucleotyde (ddNTP)

• Buffer• DNA polymerase• dNTPs• Labeled primer• Target DNA

ddGTP ddATP

ddTTPddCTP

Link: http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/Animations/DNA/WTDV026689.htm

*TTAAGTACATACCTAGTACCACTATATAATG

*TAAGTACATACCTAGTACCACTATATAATG

*TACATACCTAGTACCACTATATAATG

*TACCTAGTACCACTATATAATG

*TAGTACCACTATATAATG

*ACGCTTAAGTACATACCTAGTACCACTATATAATG

*AAGTACATACCTAGTACCACTATATAATG

*AGTACATACCTAGTACCACTATATAATG

*ATACCTAGTACCACTATATAATG

*ACCTAGTACCACTATATAATG

*AGTACCACTATATAATG

*GCTTAAGTACATACCTAGTACCACTATATAATG

*GTACATACCTAGTACCACTATATAATG

*GTACCACTATATAATG

*CGCTTAAGTACATACCTAGTACCACTATATAATG

*CATACCTAGTACCACTATATAATG

*CCTAGTACCACTATATAATG

*CTAGTACCACTATATAATG

G A C T

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

Allele A

Allele a

A

T

C

G

A

T

T

A

T

A

C

G

G

C

G

C

A

T

A

T

T

A

T

A

C

G

A

T

T

A

G

C

T

A

A

T

C

G

C

G

G

C

A

T

T

A

A

T

C

G

A

T

T

A

T

A

C

G

G

C

G

C

A

T

A

T

T

A

G

C

C

G

A

T

T

A

G

C

T

A

A

T

C

G

C

G

G

C

A

T

T

A

A a aa A aa A

Ind 1 Ind 2 Ind 5Ind 3 Ind 4 Ind 8Ind 6 Ind 7

Allele A

Allele a

A

T

C

G

A

T

T

A

T

A

C

G

G

C

G

C

A

T

A

T

T

A

T

A

C

G

A

T

T

A

G

C

T

A

A

T

C

G

C

G

G

C

A

T

T

A

A

T

C

G

A

T

T

A

T

A

C

G

G

C

G

C

A

T

A

T

T

A

G

C

C

G

A

T

T

A

G

C

T

A

A

T

C

G

C

G

G

C

A

T

T

A

A a aa A aa A

Ind 1 Ind 2 Ind 5Ind 3 Ind 4 Ind 8Ind 6 Ind 7

Labeled 3’ TGGCTAGCT 5’Probe 3’ TGGCTAGCT 5’ |||||||||Target 1 5’-CCTAACCGATCGACTGAC-3’ 2 5’-GGATTGGCTAGCTGACTG-3’

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

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