Download MALBAC Single Cell WGA kit FAQs

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MALBAC Single Cell WGA kit FAQs
1.
Q: What is MALBAC?
A: Multiple Annealing and Looping Based Amplification Cycles (MALBAC) is a quasilinear
whole genome amplification method. Unlike conventional DNA amplification methods that are
non-linear or exponential (in each cycle, DNA copied can serve as template for subsequent cycles),
MALBAC utilizes carefully-designed primers and engineered enzymes, which allow amplicons to have
complementary ends and therefore to loop, preventing DNA from being copied exponentially. This
results in preferential amplification of the original genomic DNA and therefore reduces amplification
bias. MALBAC generates more even representation of the original whole genome compared with other
amplification methods such as MDA or DOP-PCR from a single cell.
2.
Q: What applications are recommended for MALBAC single cell WGA Kit?
A: The purified products can be used on various analytical platforms such as Real-Time qPCR,
microarray and next generation sequencing (NGS). The MALBAC products of various biomedical
samples, such as laser capture microdissection (LCM), circulating tumor cells (CTC), cleavage or
blastocyst-stage embryos, fetal nucleated red blood cells, have been successfully used for analyzing
single nucleotide variations (SNVs), copy number variations (CNVs), structural variations (SVs) etc.,
with the purposes of performing pre-implantation genetic screening and diagnosis (PGS/PGD) on in
vitro fertilization (IVF)-embryos, non-invasive prenatal testing (NIPT), tumor liquid biopsy etc.
3.
Q: Can MALBAC be used for purposes of pre-implantation genetic diagnosis (PGD) or
pre-implantation genomic screening (PGS)?
A: Yes, MALBAC is especially suited for performing PGD/PGS, on both single gene diseases, as well as
comprehensive screening of all 24 chromosomes. MALBAC also enables detecting point mutations,
linkage analysis using SNP/STR, and comprehensive chromosome screening using the same biopsy
sample. Researchers have also validated the MALBAC-NGS protocol for PGS purpose by comparing
with array CGH and SNP array (Huang et al. Fertil Steril. 2014)
4.
Q: Can the product of the MALBAC WGA Kit be analyzed on a NGS platform?
A: Yes, the MALBAC amplification products have been tested and validated on next generation
sequencing (NGS) platforms such as Illumina HiSeq2500, MiSeq, NextSeq 500, and Life Technology
platforms such as PGM, Ion proton, etc. You would need to first purify the WGA products before
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performing library constructions for NGS following protocols provided by the manufacturer. We
recommend using QIAquick PCR Purification Kit for purifying the MALBAC product. (QIAGEN, Cat. #.
28104 (50rxns), Cat. #. 28106 (250rxns) ).
5.
Q: How can I determine the quality of my amplified products?
A: After the MALBAC amplification reactions are completed, 5 ul of the WGA reaction products can be
analyzed by electrophoresis (1.0% agarose gel).The majority of products should be between 300 bp
and 2000 bp in length. The total yield per MALBAC reaction processed with standard protocol should
be ~2-4 microgram measured by NanoDrop spectrophotometer.
6.
Q: Why did I get no amplified products?
A:
Possible Causes
Sample
loss
during
collection
Solutions
cell
Redo
the
cell
collection
process,
avoid
accidentally removal of the genetic material
Polymerase inhibitors carried over from the
starting materials can cause failure of the WGA
reaction. Cell washing is strongly recommended
to minimize non-cellular DNA contamination.
Mg2+-free, Ca2+-free PBS may be used for
Polymerase inhibitors
washing at 1 x concentration. Washing buffer
containing Mg2+, Ca2+, Mo2+ or Heparin should
be avoided. The washing buffer volume carried
over to the cell sample into the Lysis Protocol
should not exceed 1 ul for obtaining optimized
amplification efficiency.
All components should be stored at -20℃. All
enzymes and buffers should be freshly prepared
Inactive components
and mixed before use. Freeze-thaw cycles
should be avoided for all buffer and enzyme
tubes.
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7.
Q: I have had low amplification yield.
A:
Possible Causes
Solutions
Polymerase inhibitors carried over from the
starting material can sometimes cause low
amplification
strongly
efficiency.
Cell
recommended
washing
to
is
minimize
non-cellular DNA contamination. Mg2+-free,
Samples containing
Ca2+-free, PBS may be used for washing at 1 x
polymerase inhibitors
concentration.
Washing
buffer
containing
Mg2+, Ca2+, Mo2+ or Heparin should be
avoided. The washing buffer volume carried
over to the cell sample into the Lysis Protocol
should not exceed 1 ul for obtaining optimized
amplification efficiency.
Avoid
Degradation of genome DNA
inappropriate
template
storage
preparation
of
cells
processes
or
that
potentially degrade DNA.
8.
Q: Which kit is recommended for library construction on Illumina platforms?
A: We recommend using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® （Cat. # E7370 S/L）
with starting amount of > 100 ng for NGS library preparation purpose.
9.
Q: Would the single cell WGA kit be able to use FFPE preserved samples as input or does
it only work with fresh/frozen tissue?
A: The MALBAC single cell WGA kit performs well on cultured cells, white blood cells, FACS-sorted cells,
fresh or frozen tissues, and formaldehyde fixed tissues and cells. For FFPE samples, the amplification
efficiency and reproducibility is limited. Therefore, we recommend at least 100 cells used for analyzing
FFPE samples by MALBAC-WGA.
10. Q: Is there linear relation between starting quantity and yield? (Should I expect higher
yield with more starting quantities of materials?)
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A: NO. You would expect 2-4 microgram WGA products regardless of the starting amount with more
than one human cell. MALBAC WGA saturates at ~17 cycles with one cell, therefore, increasing the
starting amount would not increase total yield at such cycle number.
11. Q: How should I set up the procedure when I use different starting quantities? And is
there any difference in the protocol if I use different starting amounts of gDNA?
A: In the amplification step, we recommend using 14 cycles for 100 pg gDNA; 17 cycles for a single
flow-sorted mammalian cell; 19-21 cycles for a single chromosome. Number of cycles may need to be
optimized with other cell types or using other sample preparation procedure.
12. Q: Why are my negative controls bright?
A:
There are two possibilities, DNA contaminations and/or primer-primer amplification:

DNA contaminations: Please make sure all tubes and pipette tips are sterile when performing all
steps. Wear dedicated gloves and lab gowns. Preparation of the WGA reaction and processing
after WGA should always be performed in separation rooms to avoid backward contamination
from the amplified products.

Primer-Primer amplification: Random primer-primer hybridization and amplification start at room
temperature. So the reaction mix should be freshly prepared, and after mixing the pre-AMP buffer
and enzyme mix, the pre-amp reaction mix should be placed on ice (not in room temperature!).
13. Q: How can I increase total yield?
A: Scale up the reaction volume accordingly. Increasing cycle number would not increase yield if the
WGA reaction is already at saturation. For example, if ~10 ug of amplified DNA is demanded, an
option would be after performing the cell lysis step, split the lysis buffer into four PCR tubes and
perform the MALBAC amplification in each tube following the pre-amp and amplification protocol
provided.
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