Genetic Fingerprinting

DNA fingerprinting

In the R & D sector:

-involved mostly in helping to identify inherited disorders.

In forensics:

-identification of possible suspects involved in offences.

-determining the maternity or paternity of an individual.

Introduction

► DNA profiling developed by A. J. Jefferys in 1985

► Humans have most of their DNA in common

Hair, nails, saliva, blood, semen etc.

DNA fingerprinting utilizes small differences called “mini-satellites”

• 10-100 base pairs throughout the human genome which differ

• contain minute differences which make each person unique

O

OH

OCH 2 P O

O

O -

NH 2

N

N

O

OH

OCH 2 P O

O

O

-

-

N

N N H

N

O

NH 2

O

OH

OCH 2 P O

O

O

-

-

NH 2

N

N O

O

OH

OCH 2 P O

O

O

-

-

H 3 C N H

N O

O

A deoxyadenosine 5’ phosphate

G deoxyguanosine 5’ phosphate

T deoxythymidine 5’ phosphate

C deoxyctyosine 5’ phosphate

O OCH 2 P O

O

O -

NH 2

N

N

O OCH 2 P O

O

O -

N

N N H

N

O

NH 2

O OCH 2 P O

O

O -

NH 2

N

N O

O

OH

OCH 2 P O

O

O

-

-

H 3 C N H

N O

O

A

C

G

T

3’-5’ phosphodiester linkages

O OCH 2 P O

O

O -

NH 2

N

N

O OCH 2 P O

O

O -

N

N N H

N

O

NH 2

O OCH 2 P O

O

O -

NH 2

N

N O

O

OH

OCH 2 P O

O

O

-

-

H 3 C N H

N O

O

A

C

G

T

3’-5’ phosphodiester linkages

5’ end

3’ end

DNA Fingerprinting

Techniques used to distinguish between DNA of individuals:

Restricted fragment length polymorphism (RFLP)

Short tandem repeats (STR)

(DNA sequencing, polymerase chain reaction)

Two common techniques for separation and identification:

Gel Electrophoresis

Capillary Electrophoresis

DNA Samples

• Most common DNA sample are from blood or saliva, but any fluid or tissue containing DNA is suitable.

• A reference sample can be extracted using a bucal swab.

Restricted fragment length polymorphism (RFLP)

• still used but slowly replaced by more sensitive and accurate methods.

• involves fragmenting DNA with restriction enzymes.

• Restriction enzymes may be harvested from bacteria, and used specifically for DNA dissection.

Restriction Endonucleases

• Example: TaqI (Thermus aquaticus)

• Derived from hot springs bacteria

• Allows for cleavage of double stranded DNA at the phosphodiester bond

• Restriction enzymes cleave sequences which contain certain base-pairs

Resulting in “sticky” and “blunt” end fragments

Depiction of a sticky end splice

Depiction of a blunt end splice

Restriction fragments lengths are distinct and measurable A small fraction varies from person to person Variable fragments are termed Restriction Fragment Length Polymorphs (RFLP’s)

Examples of commonly used restriction enzymes

10

Gel electrophoresis

• separates the fragments on agarose gel (long DNA) or polyacrylamide gel (short DNA).

• DNA fragments can count from 300 to 10000 base pairs.

• on gel: negatively charged DNA fragments migrate to the positive end.

• DNA ladder can be compared to others.

Detection: Southern Blot

• gel soaked in a alkaline solution to denature DNA.

• denatured DNA blotted onto a nitrocellulose/nylon membrane.

• Incubation of the membrane (nitrocellulose) or exposition to UV light (nylon) for hybridization with a fluorescent or radioactive probe.

• DNA detected by absorbance measurements or on an X-ray film (radioactivity/fluorescence).

http://science.howstuffworks.com/dna-evidence.htm/printable

http://science.howstuffworks.com/dna-evidence.htm

• Probing (Hybridization)

Uses labeled single stranded DNA

This anneals to DNA which was separated

Usually 32P or bioluminescence probes

Why RLFP is becoming obsolete…

• RFLP is a qualitative approach.

• large amounts of DNA are required

• DNA tends to degrade due to harsch experimental conditions.

• Gel electrophoresis long to run.

Short tandem repeats (STR)

• STR: repeated sequences of 3-5 base pairs (loci) which can be identified in a known database.

• useful in DNA analysis because they show great variability among individuals.

• method yielding error rate of about 1 in 1029.

• does not require very much DNA, can be coupled with PCR.

• STR technology : evaluates specific polymorphic regions (loci) that are found on DNA.

•In the US, the FBI has chosen 13 specific STR loci as standard. All forensic laboratories can then establish uniform DNA databases and share forensic information.

• the likelihood that any two individuals (except identical twins) have the same 13-loci DNA profile can be as high as 1 in 1 billion or greater.

• extraction of nuclear DNA from the cells

• PCR amplification of the specific polymorphic regions

• analysis of these DNA regions by real time PCR or CE

• Determination of the number of repeats of the STR sequence in question

• CE determination use fluorescent dyes

STR Analysis

STR approach involving RFLP

DNA sequencing: the Sanger method

Also called “dideoxy”, or “termination” method (inventor Frederick Sanger, 1980 Nobel prize in Chemistry). “dideoxy”: the technique uses synthetic nucleotides lacking OH at the 3′ carbon atom. A dideoxynucleotide, when added to the growing DNA strand, stops elongation because there is no 3′ -OH for the next nucleotide to be attached to.

O OCH 2 P O

O

O -

NH 2

N

N

O OCH 2 P O

O

O -

N

N N H

N

O

NH 2

O OCH 2 P O

O

O -

NH 2

N

N O

O

OH

OCH 2 P O

O

O

-

-

H 3 C N H

N O

O

A

C

G

T

O OCH 2 P O

O

O -

NH 2

N

N

O OCH 2 P O

O

O -

N

N N H

N

O

NH 2

O OCH 2 P O

O

O -

NH 2

N

N O

O OCH 2 P O

O

O

-

-

H 3 C N H

N O

O

3’ end is hydroxylated = continuation 3’ end is deoxy = stop

Procedure The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with: • a mixture of all four normal (deoxy) nucleotides triphosphates in ample quantities

•dATP •dGTP •dCTP •dTTP

• a mixture of all four dideoxynucleotide triphosphates, each present in limited quantities and each labeled with a "tag" that fluoresces a different :

•ddATP •ddGTP •ddCTP •ddTTP

• DNA polymerase I

Because all 4 normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide instead of the normal deoxynucleotide. If the ratio of normal to dideoxy nucleotides is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

At the end of incubation, the chains are separated according to length. A difference of one nucleotide is enough to separate strands from each other. Each dideoxynucleotide fluoresces at a different λ when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html

DNA sequence of 455 nucleotides of the lysU gene of E. coli

Sequencing DNA using the ion torrentTM method

During the polymerisation of DNA, release of H+ (3’ OH) triggers an ion sensor, which generates a current. "sequencing by synthesis“: a complementary strand is built based on the sequence of a template strand.

► A microwell containing a template DNA strand to be sequenced is flooded with a single species of deoxyribonucleotide triphosphate (dNTP).

► If the introduced dNTP is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand.

► This causes the release of a hydrogen ion that triggers an ion sensor, which indicates that a reaction has occurred.

► If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

http://www.chromosomechronicles.com/2011/04/12/ion-torrent-the-dark-side-of-dna-sequencing/

Michael Glen Becker, U of M, 2013

Polymerase Chain Reaction • Developer: Kary B. Mullis

Nobel Prize, 1993

• Goal:

To clone target DNA sequences to have more for analysis

• Excellent for small amounts of DNA

PCR can “amplify” small, degraded DNA samples, or restriction fragments

• Uses Taq Polymerase

This enzyme is stable at high temperatures, as needed to denature DNA

http://cropandsoil.oregonstate.edu/classes/css430/Pics/pcr.swf

• Three main steps:

• Denaturation of DNA at about 90-960C

• Hybridization-annealing

• Taq polymerase reproduces the single strands into double strands

• The process is repeated several times to obtain a sufficient amount of DNA for analysis.

Large DNA chain from sample

Segment of interest to be copied and tested

Primer template

Primer template

A B

C D 95oC

template 3’ 5’

complement

5’ 3’

A B

C

A B

C D

D

95oC

denaturation

template

3’ 5’

complement

5’ 3’

3’ 5’

5’ 3’

complement

template

A B

C D

3’ 5’

3’

complement

template

A’

60oC Anneal 5’ Primer

5’

A B

C D

3’ 5’

3’

complement

template

A’

PCR

3’ 5’

B

C D

3’

complement

A’

3’ 5’

B

95oC

denaturation

C D

3’

complement

A’

3’ 5’

B

D’ 60oC Anneal 3’ Primer

A’ B’

PCR D’

3’ 5’

C

D’ C

C’ B’ A’