Summer Research Fellowship Programme of India's Science Academies 2017
Genetic monitoring of inbred mice by analyzing
microsatellite markers of chromosome no. 7
S Laxmi Priya
Bishop Heber College, Tiruchirappalli, Tamil Nadu
Guided by
Perumal Nagarajan
National Institute of Immunology, New Delhi
Objective
To isolate the genomic DNA of inbred mice by salted out method and NaOH-EDTA
method.
Qualitative and quantitative analysis of genomic DNA of inbred mice.
Genotyping of 11 inbred mice strains by microsatellite markers of chromosome no.7.
1. Introduction
Genetic monitoring has been developed as a testing system to assure the genetic purity of
laboratory animals and to detect the contamination by checking various gene loci or other
polymorphic locus in genome. It is an essential component of mouse colony management and
historically, has been carried out by use of immunological and biochemical markers (Nomura
et al., 1984)
Microsatellites (SSR simple sequence repeats, STR short tandem repeats, SSLP simple
sequence length polymorphism, VNTR variable number of tandem repeats) are the class of
repetitive DNA sequences present in all living organisms. Particular characteristics of
microsatellites, such as their presence in the genomes of all living organisms, high level of
allelic variation, co-dominant mode of inheritance and potential for automated analysis make
them an excellent tool for a number of approaches like genotyping, mapping and positional
cloning of genes (Monika et al., 2004).
Polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a
single or a few copies of a piece of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence. Polymerase chain reaction was
developed in 1984 by the American biochemist, Kary Mullis. Mullis received the Nobel Prize
and the Japan Prize for developing PCR in 1993 (Bartell et al., 2003)
Fig. 1. (ab) Pima-96 PCR machine.
Inbreed strains are mouse strainsthat has been maintained by sibling (sister × brother) matting
for 20 or more consecutive generations. Except for the sex difference, mice of an inbred strain
are as genetically alike as possible, being homozygous at virtually all of their loci. An inbred
strain has a unique set of characteristics that sets it apart from all other inbred strains. Many
traits do not vary from generation to generation. These traits enable them to respond to
experimental treatment with high uniformity. Researchers around the world can compare their
data with each other because they have used inbred animals (Xiaojuan et al., 2007).
In this mini project I have genotyped 11 different inbred mouse stains that are commonly used
for biomedical research -FVB/J, C3He/J, CBA/CaJ, Balb/cJ, CBA/N, C3He/Ouj, C57BL/6J,
SJL/J, 129/svJ, C57BL/10J, Balb/bJusing microsatellite markers of chromosome no. 7,
D7mit22, D7mit77, D7mit19, D7mit216, D7mit234, D7mit326 to check the purity and any
mutation or any genetic drift that are maintained for past 2 yrs in small animal facility at NII.
2. Review of literature
Mouse is a prime organism of choice for modeling human disease. Over 450 inbred strains of
mice have been described, providing a wealth of different genotypes and phenotypes for
genetic and other studies. As new strains are generated and others become extinct, it is useful
to review periodically what strains are available and how they are related to each other,
particularly in the light of available DNA polymorphism data from microsatellite and other
markers (Jon et al., 2000).
Genetic monitoring is the process of examining molecular markers to identify an animal’s
genetic makeup. This technique is commonly performed in laboratory rodents, especially mice.
Genetic monitoring is essential for quality control when breeding inbred and transgenic
animals. Any genetic contamination or genetic drift over time can alter research results and
interpretation. Therefore, accurate genotyping is necessary for obtaining quality research data.
The goal of genetic monitoring vary depending in whether the animals are outbred stocks,
inbred stains, and genetically diverse and are maintained by mating non-related animals to
retain maximum heterozygosity. Therefore the goal with genetic monitoring of outbred stocks
is to preserve the heterogeneity and prevent formation of sublines. Stains are considered inbred
if they have been maintained by 20 or more sibling mating’s. The goals of monitoring inbred
stains are to ensure a consistent genetic profile and to detect or eliminate mutations. The
purpose of monitoring genetically manipulated rodents is to identify the presence or absence
of interest as well as to verify the background stain, which may influence gene expression.
(Ryan et al., 2012).
2.1. Monitoring methods are
1. Biochemical markers and immunological techniques: Inbred mice and rats may have
variations in specific biochemical and immunological markers that can be used to
monitor genetic contamination. There are panels available to evaluate stain-specific
distribution patterns of particular markers.
2. DNA-Based Molecular techniques: There are several types of DNA-based molecular
techniques available to perform genetic monitoring. The most common techniques
include standard polymerase chain reaction (PCR) or real time PCR. Each technique
has specific use so it is important to have an understanding of the different test that
isavailable. PCR is one of the most common methods for determining genetic
variability. PCR has now replaced most of the biochemical and immunological tests
described above. Current PCR technology uses amplification of single nucleotide
polymorphism (SNPs) markers.
Microsatellite markers are simple sequence repeats within the mammalian genome that can
be used for identifying disease loci, mapping genes of interest as well as studying segregation
patterns related to meiotic non dysfunction. Different strains of mice have variable CA repeat
lengths and PCR based methods can be used to identify them, thus allowing for specific
genotypes to be assigned. (Lara et al., 2003).
PCR or polymerase chain reaction was originally developed in 1983 by the American
biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his
pioneering work. PCR is used in molecular biology to make many copies of (amplify) small
sections of DNA or a gene.it consist of following steps:
Initialization: This step is only required for DNA polymerases that require heat activation It
consists of heating the reaction chamber to a temperature of 9496°C
Denaturation: This step is the first regular cycling event and consists of heating the reaction
chamber to 9498°C This causes DNA melting, or denaturation, of the double-stranded DNA
template by breaking the hydrogen bonds between complementary bases, yielding two single-
stranded DNA molecules.
Annealing: In the next step, the reaction temperature is lowered to 5065°C. When the
temperature is lowered to enable the DNA primers to attach to the template DNA.
Extension/elongation: The temperature at this step depends on the DNA polymerase used. Taq
polymerase is thermostable it do not denature in high temperature. When the temperature is
raised and the new strand of DNA is made by the Taq polymerase enzyme. The processes of
denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required
to amplify the DNA target to millions of copies. The formula used to calculate the number of
DNA copies formed after a given number of cycles is 2
n
, where n is the number of cycles. Thus,
a reaction set for 30 cycle’s results in 2
30
copies of the original double-stranded DNA target
region.
Agarose gel electrophoresisis a method for separation and analysis of macromolecules
(DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in
clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size
independent) and in biochemistry and molecular biology to separate a mixed population
ofDNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to
separate proteins by charge (Kryndushkin et al., 2003).
2.2. Methods
2.2.1. DNA Isolation
By salted out method:
Cut 2 mm4 mm of tail of elven adult mouse inbred strains- FVB/J, C3He/J, CBA/CaJ,
Balb/cJ, CBA/N, C3He/Ouj, C57BL/6J, SJL/J, 129/svJ, C57BL/10J, Balb/BJ with the
help of a scalpel blade and added in a 1.5 mL Eppendorf tube.
To this add 300 μL digestion buffer and 35 µL proteinase K where the concentration of
proteinase K is 10 mg/mL or 0.40.8 units/sample. This is incubated in a shaker water
bath at 55C overnight.
Next day add a100 µL of 6M NaCl and shake it vigorously for few minutes.
Centrifuge at 1200013000 g for 6 min at room temperature in a cryocentrifuge.
Separate supernatant without disturbing the precipitate in a fresh tube.
Add double the volume of 95% chilled ethanol to the supernatant and mix with slight
movement.
Incubate for 30 min at 20C so that the precipitated DNA is visible.
Centrifuge at 1200013000 g for 1520 min at 4C in a cryocentrifuge.
Discard the supernatant and add 1mL of 70% ethanol.
Detach DNA precipitate by tapping so that it floats freely in the tube.
Centrifuge at 1200013000 g for 10 min at 4C in a cryocentrifuge.
Discard the supernatant and repeat the washing step with 70% ethanol once more.
Discard the supernatant and dry the pallet at 65C for 10 to 20 min in hot air oven.
Add 100 µL TE buffer and incubate in water bath at 65C for 1020 min.
Store the DNA at 20C till it is ready to use.
By NaOH EDTA method
Cut 2 mm tissue from tip of tail of eleven adult mouse inbred strains- FVB/J, C3He/J,
CBA/CaJ, Balb/CJ, CBA/N, C3He/Ouj, C57BL/6J, SJL/J, 129sv/J, C57BL/10J,
BALB/BJ with the help of a scalpel blade and add it in a 1.5mL Eppendorf tube.
Now add 75 µL of (25mM NaOH, 0.2 mM EDTA) to every eppendrof tube.
Keep at 98C in water bath or in a PCR machine for 1 h for the digestion.
Cool at room temperature.
To it add 75 µL (40mM tris HCL, 5.5 pH) to every eppendrof tube.
Centrifuge at 4000 rpm.
Take 12 µL supernatant of the sample for pcr.
Qualitative and quantitative analysis of DNA: After isolation ofDNA, quantification and
analysis of quality are necessary to ascertain the approximate quantity of DNA obtained and
the suitability of DNA sample for further analysis. This is important for many applications
including digestion of DNA by restriction enzymes or PCR amplification of target DNA. This
was done by the following techniques:
Agarose gel electrophoresis.
DNA quantification using Nanodrop.
Qualitative analysis of genomic DNA by agarose gel electrophoresis: This method of
quantification is based on the ethidium bromide fluorescent staining of DNA. Ethidium
bromide is a fluorescent dye, which intercalates between the stacked bases. The fluorescent
yield of the dye: DNA complex is much greater than the unbound dye. UV irradiation at 254
nm is absorbed by the DNA and transmitted to the dye and the bound dye itself absorbs
radiation at 302 and 366 nm. This energy is retransmitted at 590 nm, the reddish-orange region
of the visible spectrum. In case of mouse genomic DNA, the nucleic acids are
electrophoretically separated on a 0.70.8% agarose gel containing ethidium bromide at a final
g/ml. Native DNA, which migrates as a tight band of high molecular weight (presence of RNA,
and degraded/sheared DNA, if any, can be visually identified on the gel.
Procedure
Prepare a 0.8% agarose gel.
Add 1 µL of gel loading dye to 23µL of each DNA sample before loading the wells
of the gel. Addition of dye allows us to note the extent to which the samples might
have migrated during electrophoresis.
Run the submarine electrophoretic gel at 100 mV till the dye has migrated one-third of
the distance in the gel.
If you obtain one single band this means that we obtained one whole single strand of
DNA during extraction but if we obtain a ray that means there are fragments of DNA
in the sample which means too much of force has been applied during extraction of
DNA due to which the DNA fragment has broken.
Fig. 2. Qualitative analysis of geniomic DNA.
Quantitative and qualitative analysis of DNA using Nanodrop: The thermo scientific
Nanodrop 1000 spectrophotometer measures 1 µl samples with high accuracy and
reproducibility. The full spectrum (220750 nm) spectrophotometer utilizes a patented sample
retention technology that employs surface tension alone to hold the sample in place. This
eliminates the need for cumbersome cuvettes and other sample containment devices and allows
for cleanup in seconds. In addition, the Nanodrop 1000 spectrophotometer has the capability
to measure highly concentrated samples without dilution (50X higher concentration than the
samples measured by a standard cuvette spectrophotometer).
Procedure
Switch on the instrument including desktop.
After getting home page of programs, set default user and click on nucleic acid.
Set blank: Before making a sample measurement, a blank must be measured and stored
after making an initial blank measurement; a straight line will appear on the screen. For
the most consistent results, it is best to begin any measurement session with a blanking
cycle. This will assure the user that the instrument is working properly and that the
pedestal is clean. Follow the steps below to perform a blanking cycle.
Load a blank sample (1 ul of the buffer, solvent, or carrier liquid used with your
samples) onto the lower measurement pedestal and lower the sampling arm into the
‘down’ position.
When the measurement is complete, wipe the blanking buffer from both pedestals using
a laboratory wipe.
Quantify the unknown DNA.