Genetic Fingerprinting

DNA fingerprinting

In the research & development 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 consistently differ between individuals

• contain very small differences which make each person unique

O

OH

OCH 2PO

O

O-

NH2

N

N

O

OH

OCH 2PO

O

O

-

-

N

NNH

N

O

NH2

O

OH

OCH2PO

O

O

-

-

NH2

N

N O

O

OH

OCH 2PO

O

O

-

-

H3CNH

N O

O

A deoxyadenosine 5’ phosphate

G deoxyguanosine 5’ phosphate

T deoxythymidine 5’ phosphate

C deoxyctyosine 5’ phosphate

O

OCH 2PO

O

O-

NH2

N

N

O

OCH 2PO

O

O-

N

NNH

N

O

NH2

OOCH2PO

O

O-

NH2

N

N O

O

OH

OCH 2PO

O

O

-

-

H 3CNH

N O

O

A

C

G

T

3’-5’ phosphodiester linkages

O

OCH 2PO

O

O-

NH2

N

N

O

OCH 2PO

O

O-

N

NNH

N

O

NH2

OOCH2PO

O

O-

NH2

N

N O

O

OH

OCH 2PO

O

O

-

-

H 3CNH

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)

Techniques for separation and identification:

Gel Electrophoresis

Capillary Electrophoresis

Fluorescence + Next generation methods

DNA Samples

• Most common DNA samples are from blood (white blood cells) 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 (this step is still used in many approaches).

• 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 in length.

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 or labelling (Hybridization)

Uses labeled single stranded complementary DNA

This anneals to DNA which was separated

Usually 32P or bioluminescence probes

Why other methods tend to take over RLFP

• RFLP is a qualitative approach.

• large amounts of DNA are required (can use PCR to amplify)

• DNA tends to degrade due to harsch experimental conditions.

• Gel electrophoresis long to run.

Example of newer method used: real-time sequencing with Illumina.

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 if coupled to PCR.

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

• the FBI has identified 13 specific STR loci as standards. All forensic labs 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 109.

• extraction of nuclear DNA from the cells

• targeted PCR with fluorescent primer

• analysis of amplified DNA segment by RFLP

• determination of the number of repeats in the STR sequences (by size)

STR Analysis: example of method

STR approach involving RFLP

Polymerase Chain Reaction• Developer: Kary B. Mullis

Nobel Prize, 1993

• Goal:

Clone target DNA sequences to have more for analysis or other

Can “amplify” small, degraded DNA samples

Can be targeted (primers)

• Uses Taq Polymerase

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

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

• Main steps:

• Denaturation of DNA at about 90-960C (opening of double-stranded)

• START primers annealed (60oC)

• Taq polymerase makes complementary strands from START primers

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

Large DNA chain from sample

Double stranded segment to be copied and sequenced

Primersection

Primersection

A B

C

A B

C D

D

95oC

denaturation

template

3’5’

complement

5’3’

3’5’

5’3’

complement

template

A B

3’5’

template

60oCAnneal5’ Primer

D

A B

3’5’

template

PCR

3’ 5’

C D

5’

START

START

A B

3’5’

template

Denaturation 95oC3’ 5’

C D complementSTART

Anneal 3’ 5’

C D complement

5’

ASTART

PCR 3’ 5’

C D

5’ 3’

A BSTART

DC

C’ BA

DC

C’ BADC

C’ BADC

C’ BA

DC

C’ BA

Uses sequence-specific DNA probes (nucleotides labelled with a fluorescent reporter) which allow detection only after hybridization of the probe with its complementary sequence.

Or

Use of fluorescent probes that intercalate within double stranded DNA.

Goal: quantify the amount of DNA amplified.

Michael Glen Becker, U of M, 2013

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 2PO

O

O-

NH2

N

N

O

OCH 2PO

O

O-

N

NNH

N

O

NH2

OOCH2PO

O

O-

NH2

N

N O

O

OH

OCH 2PO

O

O

-

-

H 3CNH

N O

O

A

C

G

T

O

OCH 2PO

O

O-

NH2

N

N

O

OCH 2PO

O

O-

N

NNH

N

O

NH2

OOCH2PO

O

O-

NH2

N

N O

O

OCH 2PO

O

O

-

-

H 3CNH

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

Chain elongation proceeds normally until DNA polymerase inserts a dideoxynucleotide instead of the normal deoxynucleotide.

If the ratio of normal to dideoxynucleotides 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

https://www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf

As Sam gave a great talk on pyrosequencing, this technique won’t be described here.

• DNA polymerase catalyzes the incorporation of fluorescently labeled

deoxyribonucleotide triphosphates (dNTPs) into a DNA template strand

during sequential cycles of DNA synthesis.

• During each cycle, at the point of incorporation, the nucleotides are

identified by fluorophore excitation.

Four basic steps:1. Library Preparation.

• The sequencing library is prepared by random fragmentation of the DNA sample,

followed by 5′and 3′ adapter ligation.

• Adapter-ligated fragments are then PCR amplified and gel purified.

2. Cluster Generation

• The library is loaded into a flow cell where fragments are captured on a lawn of

surface-bound oligos complementary to the library adapters.

• Each fragment is then amplified into distinct, clonal clusters through bridge

amplification.

3. Sequencing

• Illumina detects single bases as they are incorporated into DNA template

strands.

• Four reversible terminator–

bound dNTPs are present

during each sequencing

cycle, but attach only

transiently.

• The result is highly accurate

base-by-base sequencing.

4. Data Analysis

The newly identified sequence reads are aligned to a reference genome.

https://www.youtube.com/watch?annotation_id=annotation_228575861&feature=iv&src_vid=womKfikWlxM&v=fCd6B5HRaZ8

Genetic fingerprinting involves different combinations of methods

e.g.: FRLP + PCR + gel electrophoresis + fluorescence labelingFRLP + fluorescence-PCR + gel electrophoresisFRLP + PCR targeted for STR + Illumina (polymerase+sequencing)PCR of segment of interest from full DNA + ion torrent …

Depending on: length and detail of DNA under investigationcostavailability of technologyreporting deadline

1. What is the Sanger method for DNA sequencing? In your answer include:

a) How complementary DNA ladders are produced;

b) How DNA segments are separated;

c) Detection and reading of sequence.

2. Explain how the polymerase chain reaction (PCR) and restricted fragment length

polymorphism (RFLP) methodologies can be applied to short tandem repeat analysis:

a) as performed nowadays with fluorescence detection;

b) as when RFLP was developed with Southern blotting.

In your answer include the role of PCR and how it can be useful in a) and b). Also,

describe how the detectable labels are incorporated in the analytical schemes.

THE ANSWERS ARE IN THE NOTES; I WILL NOT PROVIDE ANSWERS HERE.