Molecular identification of living things

Molecular Markers

Single locus markerSingle locus marker

Multi-locus markerMulti-locus marker

RFLPMicrosatellite

DNA Fingerprinting

AFLP

RAPD

PCR reactions per se are only prelude to many forms of DNA methods:

PCR-based methods

RAPD: randomly amplified polymorphic DNA

STR (= microsatellites): short tandem repeats

AFLP: amplified fragment-length polymorphisms

SNP: single nucleotide polymorphisms

DNA sequencing

RAPD is a PCR-based method which employs single primers of arbitrary nucleotide sequence with 10 nucleotides to amplify anonymous PCR fragments from genomic template DNA

What is RAPD?

RAPD technologyA B C

Genomic DNA

+

Taq polymerase

+

Arbitrary primers

A

+

Nucleotides

+

Buffer

PCR

(under relaxed conditions)

Electrophoresis

PCR

360 bp

260 bp

520 bp

520bp

260 bp

360 bp

A B C

A B C

A schematic picture of an agarose gel

Plant AMarker Plant B -

+

Plant C

Monomorphic bands

Polymorphic bands

Presens of a band, ”1” Absence of a band, ”0”

RAPD bands are treated as independent loci:

AA/Aa

aa aa aa aa aa aa aa aa aa aa aa aa aa

bb BB/Bb

BB/Bb

BB/Bb

BB/Bb

BB/Bb

BB/Bb

BB/Bb

BB/Bb

bb bb bb bb bb

CC/Cc

cc cc cc cc cc cc cc cc CC/Cc

CC/Cc

CC/Cc

CC/Cc

cc

dd DD/Dd

DD/Dd

DD/Dd

DD/Dd

DD/Dd

DD/Dd

DD/Dd

DD/Dd

DD/Dd

DD/Dd

DD/Dd

dd DD/Dd

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Locus ALocus BLocus CLocus D

1 2 3 4 5 6 7 8 9 10 11 12 13 14

RAPD bands are scored for presens ”1” and absens ”0”. Only clear, consistent and polymorphic bands are usually used to create a binary matrix for future statistical analyses

Band 1

Band 2

Band 3 Band 4

Plant A 1 0 0 1

Plant B 0 1 0 1

Plant C 1 1 1 0

Plant D 1 1 0 1

Plant E 0 1 0 1

Plant F 1 0 0 1

Plant G 1 0 1 0

A binary matrix:

Amplified Fragment Length Polymorphism (AFLP)

• Restriction endonuclease digestion of DNA

• Ligation of adaptors

• Amplification of ligated fragments

• Separation of the amplified fragments via electrophoresis and visualization

• AFLPs have stable amplification and good repeatability

Technology with elemnts of PCR-based and RFLP-based methods

Amplifies a subset of restriction fragments from a mixture of

fragments produced by digestion of genomic DNA by two

restriction endonucleases

Fragments are linked to adapters sequences so that subsequent

PCR amplifies only a subset of them (to manageable level)

Polymorphisms due to length differences of fragments

AFLP: amplified fragment length polymorphism

Restriction Fragment Length Polymorphism (RFLP)

• Genomic DNA digested with Restriction Enzymes

• DNA fragments separated via electrophoresis and transfer to nylon membrane

• Membranes exposed to probes labelled with P32 via southern hybridization

• Film exposed to X-Ray

RFLP• Restriction Fragment Length Polymorphism

• Cutting a DNA sequence using restriction enzymes into pieces specific enzymes cut specific places

Starting DNA sequence:5’-TAATTTCCGTTAGTTCAAGCGTTAGGACC3’-ATTAAAGGCAATCAAGTTCGCAATAATGG

Enzyme X5’-TTC-3”-AAG-

Enzyme X5’-TTC-3”-AAG-

5’-TAATTT3’-ATTAAA

5’-CCGTTAGTT3’-GGCAATCAA

5’-CAAGCGTTAGGACC3’-GTTCGCAATAATGG

Microsatellites

Microsatellites

What are microsatellites?– Microsatellites (also known as SSR – Simple

Sequence Repeats)

Mononucleotide SSR (A)11

AAAAAAAAAAADinucleotide SSR (GT)6

GTGTGTGTGTGTTrinucleotide SSR (CTG)4

CTGCTGCTGCTGTetranucleotide SSR (ACTC)4

ACTCACTCACTCACTC

Microsatellites

What are microsatellites?– Homozygous

…CGTAGCCTTGCATCCTTCTCTCTCTCTCTCTATCGGTACTACGTGG……CGTAGCCTTGCATCCTTCTCTCTCTCTCTCTATCGGTACTACGTGG…

5’ flanking region microsatellite locus 3’ flanking region

– Heterozygous

…CGTAGCCTTGCATCCTTCTCTCTCTCTCTCT ATCGGTACTACGTGG……CGTAGCCTTGCATCCTTCTCTCTCTCTCTCTCTCTATCGGTACTACGTGG…

Microsatellites

Where are microsatellites found?

Majority are in non-coding region

SSR Analysis

SSR: Simple Sequence Repeat or Microsatellite

• PCR based markers with 18-25 base pair primers

• SSR polymorphisms are based on no. of repeat units and are hypervariable

• SSRs have stable amplification and good repeatability

• SSR are easy to run and automate

• Most accurate molecular technique, uses genetic blackprint

(detects silent mutations and changes without length variation)

• Large selection of potential genes (mt DNA, nu DNA)

• but… expensive and time consuming and therefore only used

for limited number of genes

DNA sequencing

“PCR-mediated DNA sequencing has made some (but certainly not

all) earlier methods for generating molecular markers, especially

for phylogenetic purposes, nearly obsolete” (Avise, 2004)

In recent years, DNA sequence information has increased

explosively: by the beginning of 2003 nearly 25 million sequences

representing 115 000 taxa in GanBank!

DNA sequencing

Amplification of a gene fragment

by polymerase-chain-reaction

(PCR) with specific primer

combinations

DNA sequencing

Primers

Common mtDNA genes:

- 12S rRNA

16S rRNA (structural)

- cytochrome oxidase I,

cytochrome-b (coding)

DNA sequencing

Purification and

Sequencing

DNA sequencing

Phylogenetic reconstruction

• distance methods

• parsimony methods

• likelihood methods

DNA sequencing