Professional Documents
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Course Coordinator
Prof.Dr. Mohamed M.H. El-Defrawy
Assiut University, Faculty of Agriculture
Genetics Department, Biotechnology Lab., Assiut 71516, Egypt.
Office: +2088-2412743 Fax: +2088-2412743
Cellular phone (mobile): +20164016202
email: mmheldefrawy@yahoo.com, efrawy@aun.edu.eg
mmheldefrawy@gmail.com
Gontributors
Present address:
CIMO Research Fellow
Plant Biology (Biocenter 3), PO Box 65, FIN00014 UNIV. HELSINKI, FINLAND
Phone: +358-(0)-9-19167790
Facsimle +358-(0)-9-19157788
Former address:
Doctor of Plant Health
Dept. of Plant Science and
Biotechnology, University of Pannonia, H-8360,
Festetics 7, Keszthely, Hungary
Present address;
Ph.D Student (CICY)
Centro de investigacion cientifica de Yucatan
www.cicy.mx
Mexico - Yucatan - Merida
mobile:+5219992630144
Table of contents
DNA structure and replication
4
Genome fine structure
12
21
Types of PCRs
36
60
76
Genotyping
81
89
99
Statistical analysis
107
DNA structure
Introduction
The complete set of instructions for making an organism is called its genome.
It contains the master blueprint for all cellular structures and activities for the lifetime
of the cell or organism. Found in every nucleus of a person's many trillions of cells.
The human genome consists of tightly coiled threads of deoxyribonucleic acid
(DNA) and associated protein molecules, organized into structures called
chromosomes.
If unwound and tied together, the strands of DNA would stretch more than 5
feet but would be only 50 trillionths of an inch wide. For each organism, the
components of these slender threads encode all the information necessary for
building and maintaining life, from simple bacteria to remarkably complex human
beings. Understanding how DNA performs this function requires some knowledge of
its structure and organization.
DNA:
It's what makes you unique. It's the stuff that tells each and every one of your
body's 10 trillion cells what it's supposed to be and what it's supposed to do and
DNA Structure.
The four nitrogenous bases of DNA are arranged
along the sugar-phosphate backbone in a particular order (the
DNA sequence), encoding all genetic instructions for an
organism. Adenine (A) pairs with thymine (T), while
cytosine (C) pairs with guanine (G). The two DNA strands
are held together by weak hydrogen bonds between the bases.
A gene is a segment of a DNA molecule (ranging from fewer
than 1 thousand bases to several million), located in a
particular position on a specific chromosome, whose base
sequence contains the information necessary for protein
synthesis.
The two DNA strands are held together by weak hydrogen bonds between the bases on
each strand, forming base pairs (bp). Genome size is usually stated as the total number
of base pairs; the human genome contains roughly 3 billion bp.
(Bases)
(yeast chromosome 3)
350 Thousand
4.6 Million
5.8 Million
15 Million
50 Million
250 Million
3 Billion
Each time a cell divides into two daughter cells, its full genome is duplicated; for
humans and other complex organisms, this duplication occurs in the nucleus. During
cell division the DNA molecule unwinds and the weak bonds between the base pairs
break, allowing the strands to separate. Each strand directs the synthesis of a
complementary new strand, with free nucleotides matching up with their
complementary bases on each of the separated strands. Strict base-pairing rules are
adhered to adenine will pair only with thymine (an A-T pair, with 2 hydrogen bonds)
and cytosine with guanine (a C- G pair, with 3 hydrogen bonds). Each daughter cell
receives one old and one new DNA strand. The cells adherence to these base-pairing
rules ensures that the new strand is an exact copy of the old one.
This minimizes the incidence of errors (mutations) that may greatly affect the
resulting organism or its offspring.
Genes:
Each DNA molecule contains many genes the basic physical and functional units of
heredity. A gene is a specific sequence of nucleotide bases, whose sequences carry the
information required for constructing proteins, which provide the structural
components of cells and tissues as well as enzymes for essential biochemical
reactions.
Human genes vary widely in length, often extending over thousands of bases, but only
about 10% of the genome is known to include the protein-coding sequences (exons)
of genes. Interspersed within many genes are intron sequences, which have no known
coding function. The balance of the genome is thought to consist of other noncoding
regions (such as control sequences and intergenic regions), whose functions are
obscure. All living organisms are composed largely of proteins; humans can
synthesize at least 100,000 different kinds. Proteins are large, complex molecules
made up of long chains of subunits called amino acids. Twenty different kinds of
amino acids are usually found in proteins. Within the gene, each specific sequence of
three DNA bases (codons) directs the cells protein-synthesizing machinery to add
specific amino acids. For example, the base sequence ATG codes for the amino acid
methionine. Since 3 bases code for 1 amino acid, the protein coded by an average-
The protein-coding instructions from the genes are transmitted indirectly through
messenger ribonucleic acid (mRNA), a transient intermediary molecule similar to a
single strand of DNA. For the information within a gene to be expressed, a
complementary RNA strand is produced (a process called transcription) from the
DNA template in the nucleus. This mRNA is moved from the nucleus to the cellular
cytoplasm, where it serves as the template for protein synthesis. The cells proteinsynthesizing machinery then translates the codons into a string of amino acids that
will constitute the protein molecule for which it codes. In the laboratory, the mRNA
molecule can be isolated and used as a template to synthesize a complementary DNA
(cDNA) strand, which can then be used to locate the corresponding genes on a
chromosome map. The utility of this strategy is described in the section on physical
mapping.
10
Gene Expression. When genes are expressed, the genetic information (base sequence) on
DNA is first transcribed (copied) to a molecule of messenger RNA (mRNA) in a process
similar to DNA replication. The mRNA molecules then leave the cell nucleus and enter the
cytoplasm, where triplets of bases (codons) forming the genetic code specify the particular
amino acids that make up an individual protein. This process, called translation, is
accomplished by ribosomes (cellular components composed of proteins and another class of
ribosomal RNA, rRNA) that read the genetic code from the mRNA, and the transfer RNAs
(tRNAs) transports amino acids to the ribosome(s) for attachment to the growing protein.
11
Chromosomes
The 3 billion bp in the human genome are organized into 23 distinct,
physically separate microscopic units called chromosomes. All genes are arranged
linearly along the chromosomes. The nucleus of most human cells contains 2 sets of
chromosomes, 1 set given by each parent. Each set has 23 single chromosomes 22
autosomes and an X or Y sex chromosome. (A normal female will have a pair of X
chromosomes; a male will have an X and Y pair.) Chromosomes contain roughly
equal parts of protein and DNA; chromosomal DNA contains an average of 150
million bases. DNA molecules are among the largest molecules now known.
Chromosomes can be seen under a light microscope and, when stained with certain
dyes, reveal a pattern of light and dark bands. Differences in size and banding pattern
allow the 23 chromosomes to be distinguished from each other, an analysis called a
karyotype. A few types of major chromosomal abnormalities, including missing or
extra copies of a chromosome or gross breaks and re-joinings (translocations), can be
detected by microscopic examination; Downs syndrome, in which an individual's cells
contain a third copy of chromosome 21, is diagnosed by karyotype analysis. Most
changes in DNA, however, are too subtle to be detected by this technique and require
molecular analysis. These subtle DNA abnormalities (mutations) are responsible for
many inherited diseases such as cystic fibrosis and sickle cell anemia or may
predispose an individual to cancer, major psychiatric illnesses, and other complex
diseases.
12
Human Karyoptype
13
Alu:
GC rich
Length: ~ 280 base pairs
Location: Untranslated intronic regions
Species: Primate-specific
Methylation: Maternal
Function: mostly unknown. Do not encode
protein; LINE dependent replication; associated with some diseases (e.g. breast
cancer, hemophilia, diabetes mellitus type II).
Occasional patient
CCFDN: Only mutation identified
o
LGMD 2A
o
Mental retardation with epilepsy,
rostral ventricular enlargement
o
ACE polymorphism
o
Dystrophin-related cardiomyopathy
o Mariner (Mariner-like) elements
Flanked sides by TA dinucleotide
Length: ~80 bp
Sequence structure
o
2 perfect inverted repeat sequences
of 37 base pairs
o
Separated by six unique base pairs
(GAAAGT)
related to production of mutations in CMT
o
1A
Long interspersed sequences (LINE): The repeats are normally several thousand
base pairs in length. These sequences constitute about 21% of the human genome.
14
15
Telomeres
o Contain long arrays of TTAGGG repeats
o Repeats form nucleoprotein complex: Associate with TERF proteins.
o Repeat function
Methylation: Paternal
DNA Transposons
o Single intron-less open reading frame
o Encode transposase
o Two short inverted repeat sequences flanking the reading frame
o
Transposable repeat elements: Possible adverse effects
16
Anti-sense production
Demethylation in tumors
o Dysfunctional transposon activity
o Inappropriate gene expression
o Increased Oncogene function
Trinucleotide repeats
o
10 possible sequence motifs
o
Further functional variation depending on reading frame
o
Nomenclature:
p(CCG)n repeats
o
Frequency: Not uncommon in human genome
o
Location: Usually in 5' untranslated regions of genes
o
Repeat sizes
17
Normal: 5 to 35 copies
Intermediate range: 35 to 50
Mildly affected: 50 to 80
Imperfect
Commonly located
o
Outside of other functional domains
o
N-terminal end of proteins
o
Diseases: Poly-alanine repeat disorders
General
o
Disease types: Congenital malformations
18
gene
Tend to produce milder disorders
May not produce same disease
manifestations
Longer repeat sequences: Some disorders more
severe
Inheritance
Dominant: Usual
Normal: 6 repeats
19
20
Fig. 1 Schematic presentation of the universal structure of the rDNA region in plants. (a) The
chromosomal location of the rDNA regions. (b) Tandem arrays of the consecutive gene blocks (18S5.8S-26S). In the tandem arrays each gene block is separated by an intergenic spacer (IGS) consisting of
a 50 end and 30end external transcribed spacer (ETS). The two ETS regions are separated by a
nontranscribed region (NTS). The transcription start site (TIS) labels the start position of the 50ETS.
The small subunit (18S) and large subunit genes (5.8S and 26S) are separated by the internal transcribed
spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2).
Since its first application by Porter and Collins (1991) it has become widely used for
phylogeny reconstruction. As a part of the transcriptional unit of rDNA, the ITS is
present in virtually all organisms. The advantages of this region are: (1) biparental
inheritance, in comparison to the maternally inherited chloroplast and mitochondrial
markers; (2) easy PCR amplification, with several universal primers available for a
various kind of organisms; (3) multicopy structure; (4) moderate size allowing easy
21
22
23
24
25
26
28
Manual Techniques:
Manual hot-start, the simplest hot-start method, requires the researcher to
withhold a critical component, usually the polymerase, until the reaction has been
heated briefly at the melting temperature. Addition of the enzyme then initiates
the reaction. This method proves difficult and inconvenient to perform, especially
when processing many reactions at the same time, because the tubes must be kept
at 100 C in the PCR hot block, which serves as the working surface. This method
also increases the risk of inadvertently contaminating the reactions.
29
Chemical Modification:
31
32
Figure 3: The ReactionReady HotStart "Sweet" PCR master mix outperforms other
competing hot start enzymes. XpressRef Human Universal Reference Total RNA
(GA-004, 3 g) was converted to PCR template using the ReactionReady First
Strand cDNA Synthesis Kit. Gene-specific fragments of three different human genes
(BAX, ITGA5, IL11) were amplified by PCR from equal amounts of template using
the same primers and using either SABiosciences' HotStart "Sweet" master mix or one
of two hot start enzymes from other manufacturers, according to their respective
specifications. The enzyme in the HotStart "Sweet" master mix requires a longer
activation time than the other two enzymes. The products were characterized by
agarose gel electrophoresis.
0
1
capillary_electrophoresis
33
gel_documentation_system
Analysis of primer sequences
When designing primers for PCR, sequencing or mutagenesis it is often necessary to
make predictions about these primers, for example melting temperature (Tm) and
propensity to form dimers with itself or other primers in the reaction. The following
program will perform these calculations on any primer sequence or pair.
IDT DNA (Select Oligo Analyzer)
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
34
35
Also keep in mind that most oligonucleotide synthesis reactions are only 98%
efficient. This means that each time a base is added, only 98% of the oligos will
receive the base. This is not often critical with shorter oligos, but as length increases,
so does the probability that a primer will be missing a base. This is very important in
mutagenesis or cloning reactions. Purification by HPLC or PAGE is recommended
in some cases.
Oligonucleotide length
10 bases
20 bases
30 bases
40 bases
36
AspGluGlyPheLeuSerTyrCysTrpLeuProHisGln
GATGAAGGTTTTCTTTCTTATTGTTGGCTTCCTCATCAA
C
CT CAGC
T C
One could then select the 14 base sequence (in blue) to generate a smaller set of
degenerate oligonucleotides. Each oligonucleotide in the set would have one base
changed at a time (shown in purple below). A total of 32 unique oligonucleotides
would be generated.
TATTGTTGGCTTCC
TACTGTTGGCTTCC
TATTGCTGGCTTCC
TACTGCTGGCTTCC
etc.
When ordering degenerate oligonucleotides, you just let the company know that you
want a mixture of nucleotides added at a specific position using the code below. By
adding the mixture, oligos will incorporate one of the bases, leading to a mixture of
oligonucleotides.
Standard MixBase Definitions
A, G
C, T
A, C
G, T
C, G
A, T
A, C, T
C, G, T
A, C, G
37
38
40
(1) In intact probes, reporter fluorescence is quenched. (2) Probes and the
complementary DNA strand are hybridized and reporter fluorescence is still
quenched. (3) During PCR, the probe is degraded by the Taq polymerase and the
fluorescent reporter released.
Quantifying gene expression by traditional methods presents several problems.
Firstly, detection of mRNA on a Northern blot or PCR products on a gel or Southern
blot is time-consuming and does not allow precise quantification. Also, over the 20-40
cycles of a typical PCR, the amount of product reaches a plateau determined more by
the amount of primers in the reaction mix than by the input template/sample.
Relative concentrations of DNA present during the exponential phase of the reaction
are determined by plotting fluorescence against cycle number on a logarithmic
scale (so an exponentially increasing quantity will give a straight line). A threshold
for detection of fluorescence above background is determined. The cycle at which the
fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. The
quantity of DNA theoretically doubles every cycle during the exponential phase and
relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3 cycles earlier
than another's has 23 = 8 times more template. Since all sets of primers don't work
equally well, one has to calculate the reaction efficiency first. Thus, by using this as
the base and the cycle difference C(t) as the exponent, the precise difference in
starting template can be calculated (in previous example, if efficiency was 1.96, then
the sample would have 7.53 times more template).
Amounts of RNA or DNA are then determined by comparing the results to a standard
curve produced by real-time PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, 1:64) of
a known amount of RNA or DNA. As mentioned above, to accurately quantify gene
expression, the measured amount of RNA from the gene of interest is divided by the
amount of RNA from a housekeeping gene measured in the same sample to normalize
for possible variation in the amount and quality of RNA between different samples.
This normalization permits accurate comparison of expression of the gene of interest
between different samples, provided that the expression of the reference
(housekeeping) gene used in the normalization is very similar across all the samples.
Choosing a reference gene fulfilling this criterion is therefore of high importance, and
41
bromide can also be used for detection but its carcinogenic nature renders its use
restrictive.
43
a.This beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a
quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the
44
nine 3' bases which brings the reporter and quencher in very close proximity.
Therefore, when the reporter is excited by the appropriate light, its emission is
absorbed by the quencher and no fluorescence is detected. The pink lines represent
nucleotides that can form base pairs with the PCR product under investigation.
The PCR portion of real-time PCR is standard. Two PCR primers are used to amplify
a segment of DNA .
PCR product of interest. The two primers are show as purple arrows and the base
pairing between the two strands are shown in pink.
As the PCR continues, the newly synthesized PCR products are denatured by high
temperatures. As each strand of the product are separated, the molecular beacon also
is denatured so the hairpin structure is disrupted. As the temperatures cool for the next
round of primer annealing, the molecular beacon is capable of forming base pairs with
the appropriate strand of the PCR product (Figure 3). Any molecular beacons that do
not bind to PCR product reform the hairpin structures and thus are unable to fluoresce.
However, molecular beacons that bind to PCR product remove the ability for the
quencher to block fluorescence from the reporter dye. Therefore, as PCR product
accumulates, there is a linear increase in fluorescence.
Detection of PCR product by molecular beacon. When the beacon binds to the PCR
product, it is able to fluoresce when excited by the appropriate wavelength of light.
45
46
c. FRET HybridizationProbes
Frster resonance energy transfer (FRET), wherein an excited dye molecule transfers
its energy to a different, lower energy dye. This results in quenching of the
fluorescence from the first (donor) dye and stimulation of fluorescence from the
second (acceptor) dye, observed at longer wavelength. FRET is typically observed if
the donor and acceptor are separated by less than 10 nm and thus, it can be used to
monitor processes that result in changes in the donor-acceptor separation distance.
An interesting application of FRET is to monitor the process of RNA splicing, which
occurs in the nucleus of the cell. In humans (and many other organisms), the sequence
of a gene usually codes for a protein that would be much longer than what is actually
found when the protein is sequenced. The loss of information between DNA and
protein occurs at the RNA level by a process known as splicing. During this process,
RNA introns are
excised
out
of
the
initially
transcribed
RNA
and
the
47
48
d. Scorpion Primers
49
51
52
Figure 1. Nested PCR strategy. Segment of DNA with dots representing nondiscript
DNA sequence of unspecified length. The double lines represent a large distance
between the portion of DNA illustrated in this figure. The portions of DNA shown
53
Figure 2. The first pair of PCR primers (blue with arrows) bind to the outer pair of
primer binding sites and amplify all the DNA in between these two sites.
Figure 3. PCR product after the first round of amiplificaiton. Notice that the bases
outside the PCR primer pair are not present in the product.
Figure 4. Second pair of nested primers (red with arrows) bind to the first PCR
product. The binding sites for the second pair of primers are a few bases "internal" to
the first primer binding sites.
Figure 5. Final PCR product after second round of PCR. The length of the product is
defined by the location of the internal primer binding sites.
When a complete genome sequence is known, it is easier to be sure you will not
amplify the wrong locus but since very few of the world's genomes have been
sequenced completely, nested primers will continue to be an important control for
many experiments.
54
55
Limitations of RAPD
Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish
whether a DNA segment is amplified from a locus that is heterozygous (1
copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as
different-sized DNA segments amplified from the same locus, are detected
only rarely.
Mismatches between the primer and the template may result in the total
absence of PCR product as well as in a merely decreased amount of the
product. Thus, the RAPD results can be difficult to interpret.
New longer and specific primers are designed for the DNA sequence, which is
called the Sequenced Characterized Amplified Region Marker (SCAR).
RAPD Protocol
56
Components
Stock Concentration
Final Concentration
Vol/Rxn
dNTPs
100 mM
0.8 mM
0.2 l
10x
1x
2.5 l
MgCl2
Taq
50 mM
2.5 mM
1.25 l
5 u/l
1 u/rxn
0.2 l
Primer
10 M
0.4 M
1.0 l
dH2O
17.35 l
DNA
2 ng/l
5 ng/l
25 l
Agarose, TBE (or TAE) buffer, Ethidium Bromide, gel loading dye,
into a glass beaker or flask and add 100ml 0.5X TBE buffer (or TAE).
Microwave (or stir on a hot plate) until agarose is dissolved and solution is
clear.
57
Pour 50 C gel solution into tray to a depth of about 5mm. Allow the gel to
chamber, and cover (just until wells are submerged) with electrophoresis buffer
(the same buffer used to prepare the agarose: TBE (or TAE) buffer). Fill the
electrode tank also with 0.5X TBE buffer.
To the RAPD sample from refrigerator, add 1l of 6% gel loading dye for
every 5 l of DNA solution. Mix well. Load 15-20l of DNA per well. Load also
the DNA size standards 10 l of 1 Kb DNA Ladder (marker) alongside RAPD
reactions.
58
DNA amplification
Place PCR tubes in a thermal cycler. Amplify using the following temperature profile:
Temperature (C)
94
94
33-37
72
72
4
Time
Steps
2-5 min
60-30sec
20-30 min
0.5-2 min
5 min
59
2 for 41 cycles
Final Extension
Final hold
Capillary electrophoresis
PCR conditions must be optimized and this is normally achieved by titrating the
magnesium-, template-, primer-, dNTP- and Taq polymerase concentration, Hot Start
PCR, Touch-down PCR, adding detergents, reducing the PCR cycles or by
gradually increasing the annealing temperature. The selection of the annealing
temperature is possibly the most critical component for optimizing the specificity of a
PCR reaction. In most cases, this temperature must be empirically tested. The PCR is
normally started at 5C below the calculated temperature of the primer melting point
(Tm). However, the possible formation of unspecific secondary bands shows that the
optimum temperature is often much higher than the calculated temperature (>12C).
Further PCR reactions with gradually increasing temperatures are required until the
most stringent conditions have been found. When a standard PCR cycler is used, this
method is the most time-intensive optimization strategy. The gradient PCR now
enables rapid testing of the optimum temperature conditions on one block and in one
experiment. During the PCR, a temperature gradient, which can be programmed
between 1C and 20C, is built up across the thermoblock. This allows the most
stringent parameters for every primer set to be calculated with the aid of only one
single PCR reaction.
60
gel documentation_system
61
62
63
C (g/ml)= A260/0.027
C (g/ml)= A260/0.020
64
stranded DNA, ~37 g/ml single-stranded DNA, or ~40 g/ml single-stranded RNA
(adapted from Applied Biosystems, 1987).
For oligonucleotides: Concentrations are calculated in the more convenient units of
pmol/l. The base composition of the oligonucleotide has significant effects on
absorbance,
because the total absorbance is the sum of the individual contributions of each base
(Table A.3D.1).
3. Use the A260/A280 ratio and readings at A230 and A325 to estimate the purity of the
nucleic acid sample.
Table A.3D.1 Molar Extinction Coefficients of DNA Basesa
1M260 nm
Base
Adenine
Cytosine
Guanosine
Thymine
15,200
7,050
12,010
8,400
Ratios of 1.8 to 1.9 and 1.9 to 2.0 indicate highly purified preparations of DNA
and RNA, respectively. Contaminants that absorb at 280 nm (e.g., protein) will lower this
ratio.
Absorbance at 230 nm reflects contamination of the sample by phenol or urea, whereas
absorbance at 325 nm suggests contamination by particulates and dirty cuvettes. Light
65
0.01
0.28
0.56
0.30
2.0
28
aTypical absorbancy readings of highly purified calf thymus DNA suspended in 1X TNE buffer. The concentration of DNA was
nominally 25 g/ml.
66
68
To create the low-range standard curve, adjust the fluorometer gain to accommodate the
lower fluorescence signals.
4. Add 1.0 ml of the aqueous working solution of PicoGreen reagent (prepared in step1)
to each cuvette. Mix well and incubate for 2 to 5 min at room temperature, protected from
light.
5. After incubation, measure sample fluorescence using a spectrofluorometer or
fluorescence microplate reader and standard fluorescein wavelengths (excitation ~480
nm, emission ~520 nm). To ensure that the sample readings remain in the detection
range of the fluorometer, set the instruments gain so that the sample containing the
highest DNA concentration yields a fluorescence intensity near the fluorometers
maximum. To minimize photobleaching effects, keep the time for fluorescence
measurement constant for all samples.
6. Subtract the fluorescence value of the reagent blank from that of each of the samples.
Use corrected data to generate a standard curve of fluorescence versus DNA
concentration (see Fig. A.3D.1). Analyze samples
71
7. Add 1.0 ml of the aqueous working solution of the PicoGreen reagent (prepared in step
1) to each sample. Incubate 2 to 5 min at room temperature, protected from light.
72
The traditional method for determining the amount of DNA in solution is by measuring
absorbance at 260 nm. Because many potential contaminants of DNA and RNA
preparations also absorb in the UV range, absorption spectroscopy is a reliable method to
assess both the purity of a preparation and the quantity of DNA or RNA present.
Absorption spectroscopy does have serious limitations. Relatively large amounts of DNA
are required to get accurate readingsfor example, 500 ng/ml DNA is equivalent to only
0.01 A260 units. Furthermore, the method cannot discriminate between RNA and DNA,
and UV-absorbing contaminants such as protein will cause discrepancies. The assay
using Hoechst 33258 dye (Alternate Protocol 1) is the only procedure in common use that
is specific for DNA (i.e., it does not measure RNA). This assay is the method of choice
for rapid measurement of low quantities of DNA, with a detection limit of ~1 ng DNA.
Concentrations of DNA in both crude cell lysates and purified preparations can be
quantified (Labarca and Paigen, 1980). Because the assay quantifies a broad range of
DNA concentrations from 10 ng/ml to 15 g/mlit is useful for the measurement of
both small and large amounts of DNA (e.g., in verifying DNA concentrations prior to
performing electrophoretic separations and Southern blots). The Hoechst 33258 assay is
also useful for measuring products of the polymerase chain reaction (PCR) synthesis.
Upon binding to DNA, the fluorescence characteristics of Hoechst 33258 change
dramatically, showing a large increase in emission at ~458 nm. Hoechst 33258 is
nonintercalating and apparently binds to the minor groove of the DNA, with a marked
preference for AT sequences (Portugal and Waring, 1988). The fluorochrome 4,6diamidino-2-phenylindole (DAPI; Daxhelet et al., 1989) is also appropriate for DNA
74
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79
Troubleshooting
Common problems encountered in agarose gel electrophoresis are described
below, along with several possible causes.
Poor resolution of DNA fragments. The most frequent cause of poor DNA
resolution is improper choice of agarose concentration. Low percentage agarose gels
should be used to resolve high-molecular-weight DNA fragments and high percentage
80
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SNPs or IN
DE
Ls can
create
or
abolish
restriction
endonuclease (RE) recognition sites, thus affecting quantities and length of DNA
fragments resulting from RE digestion.
Genotyping
Developing RFLP probes
83
Southern blots of the inserts can be probed with total sheared DNA to select clones
that hybridize to single- and low-copy sequences.
The probes are screened for RFLPs using genomic DNA of different genotypes
digested with restriction endonucleases. Typically, in species with moderate to high
polymorphism rates, two to four restriction endonucleases are used such
as EcoRI, EcoRV, and HindIII. In species with low polymorphism rates, additional
restriction endonucleases can be tested to increase the chance of finding
polymorphism.
PCR-RFLP
Isolation of sufficient DNA for RFLP analysis is time consuming and labor intensive.
However, PCR can be used to amplify very small amounts of DNA, usually in 2-3
hours, to the levels required for RFLP analysis. Therefore, more samples can be
analyzed in a shorter time. An alternative name for the technique is Cleaved
Amplified Polymorphic Sequence (CAPS) assay.
84
85
86
87
90
Required reagents:
27 g of Tris base
dH2O 10mL
91
dextrose medium. Collected yeast cells were ruptured by using smashed slide
covers in the presence of liquid nitrogen while using a mortar and a pestle.
2)
Samples were then removed from the water-bath and cooled to room
Supernatant was poured and pellets were washed with cold 70%
ethanol (v/v) and centrifuged at 10000 rpm for 2 minutes at 4C. The latter
step was repeated twice, and then pellets were dried (under vacuum) at 37C
incubator (under vacuum) or left overnight at room temperature.
7)
0.5mM EDTA, (pH 8.4)). Dissolved pellets were then treated with 3l RNase
A (10 mg/ml) and incubated at 37C for 30 minutes (with occasional gentle
mixing).
8)
92
added to a 1.5 ml Eppendorf tube, mixed gently then centrifuged at 1000 rpm
for 5minutes at 20C.
10)
two volumes of cold ethanol were added to it and 1/10 volume of sodium
acetate (3M) was added to the previous mixture, mixed and left for one hour
in a freezer (-20C).
11)
Ethanol was then removed and pellets were left to dry (under vacuum)
93
94
(2nd step),
3.
Procedures:
1.
Transfer 300 l of the whole blood into a clean 1.5 ml tube.
2.
Add 900 l of RBCs lysis solution.
3.
Incubate at room temperature for 10 minutes with occasional inversion.
4.
Centrifuge at 12000 rpm for 30-60 seconds to collect the WBCs.
5.
Pour off the supernatant but leave behind about 20 l residual liquid.
6.
Add 300 l white blood cells lysis solution to resuspend pellet-pipette
up/down to lyse cells.
7.
Invert several times, then add 100 l of protein precipitation solution.
8.
Whirly mix for 20 seconds.
9.
Centrifuge at 6000 rpm for 3 minutes.
10.
Pour supernatant into a clean tube, and then add equal volume of
isopropanol to precipitate the DNA.
11.
Collect the DNA by centrifugation at 6000 rpm for 5 minutes.
12.
Gently pour off the supernatant, blot onto a paper towel.
13.
Wash once with 70 % ethanol.
14.
Decant the ethanol and leave the pellet to dry at room temperature for
10minutes.
15.
Resuspend the DNA pellet in 100 l of TE buffer.
95
16.
Conversions
Micrograms (g) = 106 grams
Micro liters (l) = 106 liters
)Milligrams (mg) = 103 grams (g
)Milligrams per liter (mg/liter) = 1 part per million (ppm
Milliliters (ml) = 103 liters
Nanogram (ng) = 10-9 grams
Picomole (pM) = 10-12 Mole
Picogram = 10-12 gram
96
97
Vaccum(.
99
Bromide .oC 50
DNA well loading DNA
) Loading Dye DNA
DNA DNA
(.
100
:
.
. :
-1
: Total Protein
.
Ployachrylamid gel Agarose Gel Band pattern
. .
101
RAPD Gel
Protein Gel
Gel Reading
-
Digital .Scanner
-
.TIFF .
Band detection
Band Matching .
.
:
-
.
-
.
-
Smiley shape .
.
Gene profiler
.
RAPD :
.www.scanalytics.com
102
:
-1
-2
Enter OK
45 .
-3
tif .
-4
) (.
-5
Analyze Mark
lanes location .
Ladder
. .
103
-6
Ladder .
-7
Ladder Standard .
104
-8
. .
-9
Edit
105
-10
. Done
.editing bands
-11
-12
-13
Match tolerance
. .
106
-14
. .
-15
1 .
File Reports .
107
-16
.
.
108
Cluster analysis
. .
Guava-5
1
1
1
1
1
1
1
Guava-4
1
1
1
1
1
0
1
Guava-2
1
1
1
1
1
0
1
Guava-3
1
0
0
1
1
0
1
Guava-1
1
1
1
1
1
1
1
Band No.
1
2
3
4
5
6
7
Cluster Analysis
Similarity Index
Dendrogram Multi Variants Statistical Package
((MVSP :
http://www.kovcomp.co.uk/mvsp
:
-1
. .
-2
Variables .
109
-3
-4
Similarity coefficient
UPGMA .Nei & li's Coefficient Advanced
Similarity Matrix .
-5
Ok Dendrogram
110
Similarity Index .
111
112