Anclaje de los grupos de ligamiento en el mapa gentico de Solanum sp mediante marcadores RFLP

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SSR assessment in potato
Daniel Andrade
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Introduction
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Natural DNA sources
Eucaryotae
Animalia Plantae Fungi Protista
Kingdoms
Prokaryotae
Archaebacteria Eubacteria
Virus
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DNA components
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DNA packing
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The plant cell
Cell wall
Membranes
Proteins
Polysaccharides
Etc.
DNA
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Plant DNA extraction
CTAB method (small scale)
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we have to break cell wall and membranes first
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Mechanical rupture of cell walls and membranes
Liquid nitrogen Freezes the leaves and allows to
break cell walls and membranes
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Incubation with CTAB at 50-65 C
CTAB dissolves membranes
CTAB NaCl 1.4 M complexes with DNA
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But we also have to protect the DNA
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Proteins that damage DNA
CTAB denatures DNAse
EDTA Captures Mg and Ca ions
Tris-HCl Holds a pH 8.0, where DNAse can work
pH 5-7
Peroxydases Polyphenoloxydases
ß-mercaptoethanol Protects DNA from peroxydases,
etc.
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Oxidants that damage DNA
Tannins Quinones Polyphenols
PVP Is an antioxydant
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Now we can isolate the DNA
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Isolating DNA
Polysaccharides Lipids Proteins
Chloroform and Isoamyl Alcohol allow to remove
polysaccharides, lipids and proteins
DNA
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Final isolation step
Isopropanol Precipitates DNA Ethanol is used
to clean DNA
DNA
RNA
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DNA solution
T1E10 dissolves and protects DNA
RNAse digests RNA
RNA
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DNA quantification and quality
Agarose electrophoresis
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What is electrophoresis?
_
Gel
Electrophoresis is a technique used to separate
molecules of different size. In this, an electric
field moves the molecules through the gel pores,
which separates one from the other while they
move according to their size
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Fago ? digested by Pst I
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Quantification of DNA by using electrophoresis
(Agarosa al 1)
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Polymerase Chain Reaction
(PCR)
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Remember
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For DNA replication is needed
1. DNA template
2. DNA polymerase
3. Nucleotides
Complementary base pairs are
1. G/C
2. A/T
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But, what is PCR for?
PCR is a technique to clone fragments of DNA in
any organism genome by using the same principals
used by the cells (DNA replication).
We need...
DNA template DNA polymerase Nucleotides Buffer Pri
mers
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To clone a DNA fragment We have to follow three
basic steps
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First step Denaturation
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The DNA is heated at 94C
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The DNA is heated at 95C Until the strands melts
open
TTAACGGGGCCCTTTAAA
TTTAAACCCGGGTTT
AATTGCCCCGGGAAATTT........AAATTTG
GGCCCAAA
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Second step Annealing
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The temperature goes down to around 54C
TTAACGGGGCCCTTTAAA
TTTAAACCCGGGTTT
AATTGCCCCGGGAAATTT........AAATTTG
GGCCCAAA
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The temperature goes down to around 54C so that
the primers can attach to the DNA template
correctly
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Third step extension
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The temperature goes up to around 72C
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The temperature goes up to around 72C because
this is the optimal temperature for taq-polymerase
TTAACGGGGCCCTTTAAA
TTTAAACCCGGGTTT
AATTGCCCCGGGAAATTT
TTTAAACCCGGGTTT
AATTGCCCCGGGAAATTT........AAATTTG
GGCCCAAA
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TTAACGGGGCCCTTTAAA
TTTAAACCCGGGTTT
3
AATTGCCCCGGGAAATTT
5
TTTAAACCCGGGTTT
5
AATTGCCCCGGGAAATTT........AAATTTG
GGCCCAAA
3
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TTAACGGGGCCCTTTAAA
TTTAAACCCGGGTTT
3
AATTGCCCCGGGAAATTT
5
TTTAAACCCGGGTTT
5
AATTGCCCCGGGAAATTT........AAATTTG
GGCCCAAA
3
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And in the end...
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We obtain the wanted fragment cloned
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But, it is not that easy
PCR takes several steps called cycles
A cycle consists of...
Denaturation 94C Annealing around
54C Extension 72C
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Sequencing DNA
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Nucleotide polymerization
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5
3
3
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How do didinucleotides work?
G ddGTP A ddATP T ddTTP C ddCTP
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The sequencing reaction
Plasmid Primer Nucleotides (dNTPs) DNA
polymerase Buffer.
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What is a microsatellite?
A microsatellite is a DNA fragment formed by
simple sequence repeat units
TCT
TCT
TCT
TCT
TCT
TCT
TCT
TCT
TCT
AGA
AGA
AGA
AGA
AGA
AGA
AGA
AGA
AGA
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These units or motives can be formed by from 1 up
to 5 base pairs
GGTA
GGTA
GGTA
GGTA
GGTA
GGTA
GGTA
CCAT
CCAT
CCAT
CCAT
CCAT
CCAT
CCAT
G
G
G
G
G
G
G
G
G
C
C
C
C
C
C
C
C
C
TC
TC
TC
TC
TC
AG
AG
AG
AG
AG
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What is a microsatellite marker like?
It is a DNA fragment containing not only the SSR
region but an extra DNA portion It needs a couple
of primers (around 20 bp.each) to be amplified
Genomic DNA
(TC)14
......CAGCCCGTAAATGTATCCATCATTAGCTTTCGATAAAGACCATC
T C TCTCT CTC TCT CTC TCTCTC T CT
CTGTGAAACCCAGTTGAATTAGAATTTTGAATTT...... ......GTC
GGGCATTTACATAGGTAGTAATCGAAAGCTATTTCTGGTAGAGAGAGAGA
GAGAGAGAGAGAGAGAGACACTTTGGGTCAACTTAATCTTAAAACTTAAA
......
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5
5
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SSR are codominant markers
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Origin of the SSR polymorphism
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SSR are all over the genome
Solanum phureja
SSR Loci
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How to score SSR markers
Peso (pb)
A
B
C
D
E
167(T)
0
0
1
0
0
164(C)
1
0
1
0
0
155(A)
0
1
0
1
0
139(T)
0
1
0
1
1
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S011- Gel I
S051- Gel III
S052 - Gel II
S069 - Gel III
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Thats all folks!