PLB161A Laboratories X to XIV
Genome Mapping
http://agronomy.ucdavis.edu/Dubcovsky/PLB161A/PLB161A_Home.htm
Jorge Dubcovsky
Table of contents
Page
X a Isolation of DNA from plants
5
X b DNA quantification
11
Lab report
16
XI a Restriction digest and electrophoresis
17
XI b Southern blot
25
Lab report
29
XII Probe preparation and hybridization
30
XII Autoradiography
40
Lab report
42
XIII PCR markers
43
Lab report
49
XIVa Molecular markers: analysis of results and mapping
50
XIVb Construction of genetic maps using MapMaker
63
Lab report
71
XV Quantitative trait loci analysis (QTLs)
72
Objective: To become acquainted with molecular techniques commonly used to
analyze the structure, function, and evolution of genomes.
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Background information:
Introduction to Genome Mapping
The molecular characterization of a genome encompasses several stages
of increasing resolution and sophistication, leading to the generation of a highresolution chromosome maps and eventually to the sequencing of the genome.
The first step to genome analysis requires the assignment of genes and
molecular markers to specific chromosomes or linkage groups. The next step
entails calculating the map distances between gene loci on a chromosome.
Finally, physical mapping and sequencing provides the highest level of resolution
in genome analysis. A fine structure map can be used to address fundamental
and practical questions. Some examples are i) the organization of functional vs.
nonfunctional DNA regions, ii) elucidation of evolutionary processes and
relationships, iii) systematic and taxonomic studies, iv) positional cloning, v)
manipulation of genes and chromosome segments in breeding programs (marker
assisted selection), etc.
Chromosome Mapping
Determining the location of a gene or molecular marker on a chromosome
map relies on special cytogenetic stocks or linkage mapping, as measured by the
recombination frequency in dihybrid and multihybrid crosses. Historically, genetic
linkage maps or chromosome maps were assembled by mapping genes with
qualitative phenotypic differences to individual loci through controlled
experimental crosses. Due to the limited number of morphological or
physiological traits present in the initial maps, distances between loci were large
and encompassed numerous unknown genes. These “gaps” were essentially
inaccessible to linkage analysis because of the lack of available phenotypes
assigned to those regions. The discovery and implementation of molecular
markers provided large numbers of additional genetic markers able to fill in the
gaps and provide a higher-resolution map in the process. A molecular marker is
a site of heterozygosity for some type of DNA variation. Such a DNA marker in
mapping analysis is analogous to a conventional heterozygous allelic pair. The
identity of the DNA is not important, but is simply used as a reference point to
arrange and order the genetic information along the length of a chromosome.
The molecular markers provide landmarks along the chromosome that facilitate
the manipulation of chromosome segments in breeding and chromosome
engineering programs.
Restriction Fragment Length Polymorphisms (RFLPs)
The utility of DNA-based molecular markers in genome mapping emerged
in the 1980's. The first such markers are known as restriction fragment length
polymorphisms (RFLPs). Such polymorphisms are simply due to the presence or
absence of a restriction enzyme recognition site or the insertion /deletion of DNA
sequences between two restriction sites. Even a single base-pair difference in
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Parent A
Parent B
the nucleotide sequence can be responsible for generating an RFLP. RFLPs are
identified by screening genomic restriction digests of the parents of a segregating
population with different restriction enzymes with cDNAs or randomly cloned
genomic fragments. Then the DNAs from the complete segregating population
are digested with the selected restriction enzyme and hybridized with the same
probe as exemplified below.
F2 segregating population
RFLP autoradiographic image
Clone: CDO348 Enzyme: Sst I
The next step after an RFLP map is available is the construction of
physical map of the organism. This is usually accomplished by cloning random
large segments (100-150kb) of the genome in artificial bacterial chromosomes
(BAC clones). Large numbers of clones are used to saturate the genome, usually
including 5-10 copies of the complete genome. For example, the BAC library for
tetraploid wheat constructed in my laboratory includes 500,000 clones.
BAC clones can be fingerprinted using different techniques and the
overlapping groups of BACs can be established (Contigs). Hybridization of the
BAC libraries with the clones used to construct the RFLP map connects the
physical and genetic maps.
These contigs can be use to select minimum set of overlapping BACs for
sequencing. The complete sequence of an organism is the final level of
resolution of genome mapping. However, it is just the beginning of an incredible
adventure in biological research in gene function, gene interactions, allelic
diversity and evolution.
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RFLP PROCEDURE
Plant Genomic DNA or cDNAs
Harvest leaf tissue
Lyophilization
Clone into vector
Dried leaf tissue
Transformation
Tissue grinding
Plasmid inserted in host
Ground leaf tissue
Mini-preps
DNA isolation
Plasmid DNA
Genomic DNA
PCR or restriction digest
Restriction digest
Isolated insert
Digested DNA
Random priming
Liquid nitrogen
Ligation
Gel electrophoresis
Labeled insert: probe
DNA fragments
separated in gel
Southern blotting
Membrane with DNA
Hybridization
Probe hybridized
to blot
Wash
Autoradiography
Result and analysis
Stripping
Probe removed from blot
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Laboratory X a
1.- Genomic DNA extraction from plants
1.1. Background information:
Plants contain three types of DNA: nuclear, mitochondrial and chloroplast
DNA. Although quite elaborate methods exist for the isolation of each type of
DNA, most experiments require only the rather simple preparation of total DNA.
All DNA preparation methods involve the removal of the cell wall and nuclear
membranes, the separation of the DNA from all other cell components, and the
protection of the DNA during the procedure from nucleases and mechanical
shearing.
The two main problems in isolating DNA from plants are the presence of
DNAase activity, which degrade the DNA, and the presence of other
macromolecules, which co-purify with, or polymerize to, the DNA during the
isolation procedure.
DNAase
Low temperatures reduce DNAse activity of DNA and therefore most of
the procedures are performed in ice or in cold centrifuges when the DNA is not
protected. High Ph in some extraction buffers also contributes to DNAase
inactivation.
The nuclease problem is reduced by removing cations such as Mg++ which
are required for nuclease activity using EDTA. This is why you store your DNA in
TE. In addition, detergent agents such as sodium dodecylsulphate (SDS) or
Sarkosyl are often used to inhibit enzyme activities, to dissolve membranes, and
to dissociate proteins from DNA to make them more accessible to degradation.
Different chemicals are used to destroy DNAses including, the addition of fresh β
mercaptoethanol to the extraction buffer to disrupt protein disulfide bonds, or
Proteinase K that digest proteins (good but expensive), or simply by a
Phenol:Chloroform extraction to degrade all proteins.
Other macromolecules
One common problem for DNA extraction in some plant species or some
tissues, is the presence of high-concentrations of polysaccharides which copurify with DNA in normal phenol-chloroform extractions. Isolation of DNA from
these species and tissues is preferably achieved by treatment with CTAB
(cetyltrimethyl ammonium bromide). The DNA is soluble in the presence of CTAB
at high salt concentration (0.7 M). A CTAB-nucleic acid precipitate will form if the
salt concentration drops below 0.4 M at room temperature. The salt
concentration of the buffer is maintained at 0.7-M and the CTAB forms
complexes with cell wall debris, other polysaccharides and proteins. This CTABprotein/polysaccharide complex is removed by chloroform extractions leaving
DNA in the aqueous phase to be ethanol precipitated.
-5-
Plant researchers often encounter undesirable macromolecules, other
than DNA, which create problems in the DNA isolation procedure. In some
plants like beans, grapes, etc, is necessary to add 5% polyvinyl polypyrrolidone
(PVPP, Sigma, P6755) to the extraction buffer to purge polyphenols. PVPP
forms complex hydrogen bonds with polyphenolic compounds that can be
separated from DNA by centrifugation 1. PVPP is usually not necessary to
isolate DNA from grasses.
1.2. General Instructions:
You will isolate DNA from bread wheat (Triticum aestivum) nulli-tetrasomic
cytogenetic stocks. Wheat is an hexaploid species (2n=42 chromosomes) and
has three genomes designated A, B, and D with seven chromosomes each. The
nulli-tetrasomics are lines of the land race Chinese Spring that have one missing
chromosome pair and a compensating homeologous chromosome pair in double
doses. For example, nulli-1A-tetra-1B, has two chromosomes 1D, four
chromosomes 1B, and no chromosome 1A (the total is still 2n=42
chromosomes). These cytogenetic stocks are very useful to determine the
chromosome location of genes and molecular markers.
Though there are 21 possible nullisomics, each group will use only 14 of
them to be able to run them in the 15-teeth combs we have for the course. We
will use 2 nullisomic-tetrasomic lines per chromosome.
1) N1BT1A
3) N2BT2D
5) N3BT3A
7) N4BT4D
9) N5AT5D
11) N6BT6D
13) N7BT7A
2) N1DT1A
4) N2DT2A
6) N3DT3B
8) N4DT4A
10) N5DT5B
12) N6DT6A
14) N7DT7B
Each mini-extraction provides approximately 10 µg of DNA that is
sufficient for RFLP analysis of single copy genes in wheat. Each group will
perform one mini-extraction per each of the 14 lines.
Important: All steps involving phenol/chloroform must be performed under the
fume hood using lab coat and gloves
DNA isolation:
1. Prepare and label your tubes using a letter for your group and numbers 1 to
14. Use double labels (top and one side).
Remember that
Correct labeling is the most critical step in genetic studies
1
Lodhi, M. A., G.-N. Ye, et al. (1994). “A simple and efficient method for DNA extraction from
grapevine cultivars and Vitis species.” Plant Molecular Biology Reporter 12: 6-13.
-6-
2. Collect 10cm long piece of young leaf material without necrotic regions. Fold
it tightly into a 1.5 ml tube.
3. Freeze in liquid Nitrogen. Grind the leave to a fine powder with the end of a
brush. Keep tissue frozen all time (you can put it back in the liquid N and
then continue grinding).
The low temperature prevents nucleases from degrading the DNA.
4. Add 500 µl of cold DNA extraction buffer to the frozen powder and mix well
with a spatula
5. Add 500 µl phenol:chloroform:isoamyl alcohol (25:24:1) in the HOOD and
shake well. Use protective glasses and gloves.
6. Centrifuge for 3 minutes at room temperature and maximum speed. Transfer
upper phase (about 500 µl) to a new tube with a P1000 pipette
7. Add 500 µl of cold Chloroform mix gently, centrifuge for 1 minute and take
supernatant (about 500 µl) to a fresh tube. Do not collect any Chloroform
8. Add 1/10 of the volume (approximately 50 µl) of 3M Sodium acetate (PH=4.8)
and one volume (approximately 500 µl) of isopropanol (2-Propanol). Mix
gently. DNA will precipitate. Centrifuge for 5 minutes.
9. Pour off supernatant carefully. Wash pellet with 500 µl of 70 % Ethyl alcohol,
Spin down and remove the ethanol with P1000. Dry pellet 5 minutes and
resuspend in 50 µl of sterile TE. Leave the tubes open in the lamina flow for
15 minutes to evaporate any residual ethanol.
10. Take 5 µl of each sample and combine it with 20 µl mixture of buffer and
restriction enzyme TaqI (this mixture will be provided= 2.5µl buffer, 0.2 µl
enzyme, 17.3 µl water). Use small PCR tubes. Place the samples at 65 C in
the PCR machine. These samples will be used next lab to test the digestibility
of the extracted DNA.
11. Store the DNAs at 4 C.
Solutions
DNA Extractio buffer (for 1 Lit.)
33 ml 30% Sarkosyl
12.1 g. Trizma base
5.8 g. NaCl
3.2 g Na 2EDTA
PH=8.5 by HCl
Phenol : Chloroform : Isoamyl alcohol
250ml redistilled equilibrated phenol
240 ml chloroform
10 ml Isoamylalcohol
-7-
1M Tris pH8.0: Dissolve 121 g Tris-Base in approximately 750 ml of distilled
water. Add concentrated HCl until desired pH is reached (approximately
49 ml for pH8.0). Bring solution to 1000 ml with distilled water. Autoclave.
0.5 M Edta pH 8.0: Dissolve 186.12 g of EDTA in approximately 750 ml of
distilled water. Add NaOH pellets to bring pH to 8.0 (approximately 20 g).
After EDTA is in solution, bring to 1000 ml with distilled water. Autoclave.
TE pH 8.0 (Autoclave):
Stock
1M Tris pH 8.0
50 ml
0.5 ml
100 ml
1.0 ml
500 ml
5.0 ml
1000 ml
10.0 ml
2000 ml
20.0 ml
0.5 M EDTA pH 8.0
0.1 ml
0.2 ml
1.0 ml
2.0 ml
4.0 ml
dd H 2O
To volume
To volume
To volume
To volume
To volume
1.3. Alternative method to separate DNA extraction from sample collection:
1.3.1. Lyophilization
The use of lyophilized tissue offers several advantages. Dry tissue can be
efficiently disrupted while the DNA is dehydrated and thus less susceptible to
shear. Since the DNA is then hydrated immediately in the presence of detergent
and EDTA, nucleolytic degradation is minimized. Finally, dry tissue can be stored
for several years with little loss of DNA quality, DNA extraction can be postponed
until the segregation of phenotypic characters is confirmed, and large number of
samples can be processed simultaneously.
The vapor pressure of the product forces the sublimation of the water
molecules from the frozen product to the condenser In this technique, a larger
amount of tissue is harvested (15-20 grams). Leaves are folded in a fiberglass
screen mesh bag along with the tag identifying the sample and placed inside
liquid nitrogen. If you are not planning to lyophilize the samples immediately,
place leaf samples at -80 C until ready to be lyophilized. Once frozen, do not
allow samples to thaw until freeze-dried!
When you transfer leaf samples to lyophilizer you need to make sure that
the lyophilizer is down to temperature (the chamber is < -60 °C) and pulling a
good vacuum (< 100 microns Hg) before loading samples. Do not overload
lyophilizer. Samples should be dry in 72 hours. Typically, fresh weight = 10X dry
weight.
Dried leaf samples may be stored in sealed plastic bags at room
temperature for a few days or, preferably, at -20 °C for several years. Before
extraction grind the frozen samples to a fine powder with a mechanical mill into a
container that can be closed airtight (Samples are stable at -20 °C for several
months).
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Liquid
Phase
Va
po
riza
tio
n
Solid
Phase
A
line
ine
on l
Fusi
Pressure
Freeze drying
B
C
Vapor
Phase
Temperature
Phase diagram
1) Prefreezing: Put leaves at –80 °C
2) Freeze drying
• A vacuum pump is used to lower the pressure of the environment
around the product
• A cold trap is used to collect moisture that leaves the frozen
product
1.4 High Molecular Weight DNA extraction methods
(Zhan et al Plant J. 7:7:175-184.)
For the construction of large insert genomic libraries like the Bacterial
Artificial Chromosome (BAC) libraries, it is important to extract DNA of very high
molecular weight. The problem is that when DNA is dissolved into water it will
immediately break into smaller pieces. To avoid this problem nuclei are isolated
and then embedded into low melting agarose in small plugs. All subsequent DNA
manipulations are done INSID E the agarose (including nuclear membrane lysis,
restriction enzyme digests, etc.)
When the plugs are completely solidified, they are transferred to a buffer
with 1mg/ml of Proteinase K (Boehringer-Mannheim). Plugs are first placed in ice
for 30 minutes to let the proteinase K reach the DNA, than incubate 24 hrs at 50
°C with shaking (these washes are repeated twice). Then plugs can be stored at
4 °C in 10:10 TE (TE with 10 times more EDTA than normal 10:1 TE).
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TE
λ
ladder 10:10
100g of tissue
Ground in liquid N2
in HB buffer
388
339
Homogenate
291
242
Nuclei isolated
194
Nuclei
145
Nuclei
embedded in
LMP agarose
Agarose plugs
97
48
Lysis and protein
degradation
HMW DNA
Pulse Field Gel conditions:
1”-40”/200v/12ºC/20h
-10-
TE
10:1
1 plug 1/2 plug
water water