苏州大学 尹斌 教授
The main goal of the “973” project that we are carrying out is to explore the biological principles governing the development and progression of leukemia, utilizing the state-of-the-art nanotechnologies which are ongoing in our laboratories. Now, we would like to share one of the approaches to dissecting genetic and genomic mechanism(s) underlying leukemogenesis. These methods have been published recently and therefore, are accessible (Bin Yin. Isolation of Genomic Insertion Sites of DNA element using Splinkerette-PCR based Procedures. Methods Mol Biol. 2011;687:25-42).
Isolation of Genomic Insertion Sites of Proviruses using Splinkerette-PCR based Procedures
Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Soochow University, Suzhou, China. Email: firstname.lastname@example.org.
The availability of whole genomic sequences provides a great framework for biologists to address a broad range of scientific questions. However, functions of most mammalian genes remain obscure. The forward genetics strategy of insertional mutagenesis uses DNA mutagens such as retroviruses and transposable elements; this strategy represents a powerful approach to functional genomics. A variety of methods to uncover insertion sites have been described. This chapter details SplinkTA-PCR and SplinkBlunt-PCR, modified from splinkerette-PCR, for mapping chromosomally the insertion sites of a murine leukemia virus that causes leukemia in the BXH-2 strain of mice. These protocols are easy to use, reliable, and efficient.
With the whole genome sequences of human and major model organisms available, biological processes can be explored in an unprecedented way – questions with significantly increased broadness and complexity are pursued on a large-scale and at a systemic level. On the wave of interest in dissection of gene functions and biological pathways, chromosome insertional strategies using DNA elements such as retroviruses and transposons represent a powerful forward genetics approach to functional genomics, among other induced or engineered mutagenesis (1-5). These DNA mutagens integrate into host genome DNA and can alter gene function. Through the identification of the chromosomal insertion sites of the mutagens, a gene can be assigned a certain phenotype-related function. Recently, insertional mutagenesis has been demonstrated to be fruitful in genome-wide analysis of cancer genes (6-13). The BXH-2 strain of mice constitutes a model of human acute myeloid leukemia (AML) which arises from infection by a murine leukemia virus (MuLV) (14). The BXH-2 MuLV not only acts as an insertional DNA mutagen to cause leukemia, but also serves as a tag for leukemia-associated genes.
A variety of methods used to uncover insertion sites have previously been described, including genomic DNA library screening (15), ligation-mediated PCR (6), inverse PCR (16), VISA technique (17), T-linker PCR (18), and single nucleotide polymorphism-based mapping (19). These methods have been adopted to generate a large amount of insertion sites; however, their inherent limitations are posed either by excessive laborious work, low cloning efficiency, restriction site-related cloning bias, or non-specific amplification. To facilitate functional genomic studies and maximize information that can be delivered from the use of insertional mutagenesis strategy, it is crucial to capture as complete a genome-wide profile of insertion sites as possible. The protocols that will be elaborated in this chapter provide a time-saving tool allowing for less laborious, less biased and more efficient mapping of insertion sites.
The splinkerette-PCR is derived from vectorette-PCR (20), also similar in principle to cassette ligation-anchored PCR (21), single-specific-primer PCR (22), rapid amplification of cDNA ends (23, 24), and rapid amplification of genomic DNA ends (25). This PCR begins with the digestion of genomic DNA with a restriction enzyme, followed by ligation of a double-stranded DNA linker, namely splinkerette, to the digested DNA. The insertion flanking sequences are then PCR amplified using a pair of primers specific to the integrated DNA and the splinkerette, respectively (26). Splinkerette-PCR features a hairpin structure present in the splinkerette which help overcome the undesired amplification, so-called “end-repair priming phenomenon”, that decreases the specificity of conventional linker mediated-PCR. Another advantage of the approach is the elimination of the requirement of circularization that can often be a problematic step in some types of PCR. Splinkerette-PCR has been used to characterize genomic integration of provirus, transposon and gene trap vector (6, 13, 27, 28). However, there is still a need for the development of more sophisticated and streamlined protocols that better accommodate the emerging large-scale insertion site mapping efforts. In line with this trend, a web-based automated analysis and mapping of insertional mutagenesis sequence data has recently become available (29).
2. SplinkTA-PCR (STA-PCR)
In our experience of cloning retroviral insertion sites, splinkerette-PCR worked better than inverse PCR; however, splinkerette-PCR mostly produced smear – there were rarely discernible discrete PCR bands for recovery and cloning. This is likely due to excessive non-specific amplification, which is consistent with other investigators’ observations (18). We also tried to clone the resulting smear PCR products for sequencing, only to find a very low percentage of positive colonies and an even lower number of specific PCR amplicons. With inverse-PCR, we observed even fewer PCR bands. In order to achieve saturation recovery of insertion sites, a more efficient approach is needed. SplinkTA-PCR is developed from splinkerette-PCR, and its strategy is illustrated in Figure 1A (30). This PCR approach starts from digestion of genomic DNA with a cocktail of restriction enzymes generating 5’ overhang ends. The digested DNA was purified, and modified at the 3’ end with addition of an adenosine, by taking advantage of the terminal non-templated extension capacity of conventional Taq DNA polymerases (31, 32). This “A”ddition step is followed by ligation to SplinkTA, a modified splinkerette linker, which is made up with Splinkerette (the hairpin oligo) and PrimerLongTA (the oligo with an extra thymidine at 3’ end). Primary PCR was then performed on the ligation products as templates using primers specific to BXH-2 MuLV and SplinkTA, respectively. In secondary PCR, templates are switched to primary PCR products, and primers to a nested pair of primers specific to the long-terminal repeats of BXH-2 MuLV and complementary to PrimerLongTA, respectively. Individual secondary PCR bands were resolved on agarose gel and subsequently recovered for sequencing.
SplinkBlunt-PCR is also modified from splinkerette-PCR (30). In this PCR method, as illustrated in Figure 1B, genomic DNA is first digested with a cocktail of restriction enzymes. Next, a biotinylated primer was annealed to the digests to drive the primer extension reaction. Pfu DNA polymerase was used to produce blunt-end double-stranded DNA fragments. The extension products were subsequently purified with streptavidin magnetic particles, prior to ligation to SplinkBlunt which was made by mixing Splinkerette with PrimerLong (the oligo without extra thymidine at the 3’ end), using the same procedure as in the STA-PCR protocol. Following ligation, the biotin-bearing DNA fragments were captured magnetically, denatured, and separated from other unbound DNA fragments. The supernatant was collected and used as templates for subsequent primary and secondary PCR which were performed similarly to what was described in STA-PCR Protocol. The downstream procedure following amplification is the same as that in STA-PCR.
STA-PCR and SplinkBlunt-PCR can consistently recover more insertion sites of BXH-2 MuLV with high specificity (for an example, see Figure 2). The high efficiency of SplinkTA-PCR may be attributed to the integration of the following improvements: 1). Use of multiple enzymes rather than a single enzyme for one digestion reaction gives rise to more readily amplifiable DNA fragments, reduces insertion cloning bias introduced by single restriction enzyme-dependent digestion, saves time and effort by alleviating the requirements of more reactions and linkers which are necessary for single enzyme-based protocols; 2). Adoption of splinkerette eliminates the end-repair priming phenomenon and thereby results in greater efficiency in ligation-mediated PCR (26); 3). Formation of the SplinkTA in STA-PCR with an extended thymidine at the end increases ligation efficiency, maximizes its compatibility with a variety of digestion fragments generated by different restriction enzymes, and decreases the likelihood of forming tandem blunt ligation products; 4). Inclusion of primer extension and purification steps in SplinkBlunt-PCR eliminates the majority of non-template DNA which would adversely affect efficiency of downstream PCR, since the magnetic beads used in the purification only bind and retain double-stranded DNA fragments formed via streptavidin-labeled primers.
When comparing between STA-PCR and SplinkBlunt-PCR, the former has fewer steps and reduced likelihood of formation of tandem ligation. SplinkBlunt-PCR is characterized by cleaner PCR gel background, and no limitation in choice of restriction enzymes to 5’ overhang-producing enzymes because both 3’ overhang and blunt DNA fragments can be primer extended. In addition, although comparable efficiency was observed for STA-PCR and SplinkBlunt-PCR in cloning of BXH-2 insertion, there are slight differences between these two methods in the recovery profile of insertions (see Figure 3).
The ability of STA-PCR and SplinkBlunt-PCR to recover insertion sites could be enhanced by digesting genomic DNA with more than one combination of restriction enzymes. We have successfully tested various combination of restriction enzymes, including: (I) Aat II, Mfe I, Nde I, (II) Hind III, Pvu II, Xho I or (III) Ase I, Bgl I, Eag I, with different resultant PIS patterns. Further facilitation of mapping insertions can be made by running through the cloning procedures for both upstream and downstream of the DNA integrated. We expect that with minor modification, these protocols can be readily adapted to identify insertion sites of other types of insertional mutagens, such as transposons, retrotransposons, etc. The techniques can also be applied to determine endpoints of genomic DNA fragments, chromosomal breakpoints involved in deletion or translocation, intron-exon junctions and gene regulatory regions.
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Tables and figures:
Table 1. Sequence (5’-> 3’) of oligonucleotides used in STA-PCR and SplinkBlunt-PCR
Figure 1 Schematic diagram of strategies for cloning genomic DNA sequence flanking a proviral insertion. (A). STA-PCR protocol. Genomic DNA (horizontal lines) is digested with a cocktail of restriction enzymes (noted as E, F and G) which have no recognition site downstream to the primers (horizontal arrow heads) used for primary and secondary PCR within proviral genome sequence (black boxes). The digestion produces 5’ overhang ends which are subsequently filled in and added an adenosine at the ends with Taq DNA polymerase. The filled-in products are ligated to SplinkTA, a modified Splinkerette with a thymidine (empty star) at the end which can exactly pair to the adenosine (filled star) at the end of extension products. Then two consecutive rounds of PCR are performed followed by PCR products cloning and sequencing. (B). SplinkBlunt-PCR protocol. Genomic DNA (horizontal lines) is digested with restriction enzymes (X, Y and Z in this case) in a similar way described in Fig.1A. After the digested double stranded DNA is denatured, proviral genome sequence specific primer, which is biotinylated at its 5’ end (filled circle), is annealed to its cognate region on single stranded DNA fragments and immediately extended with Pfu DNA polymerase to produce blunt ends which are subsequently ligated to SplinkBlunt. Those biotin-bearing DNA fragments are then purified with streptavidin magnetic particles prior to two rounds of PCR and products cloning as performed in Fig.1A (Taken from Yin, B. et al., BioTechniques 2007. © 2008 BioTechniques. Used by Permission).
Figure 2 Application of PCR-based protocols to large-scale screen of proviral insertion patterns in BXH-2 leukemias. Lanes 1 ~ 17 are leukemia samples. The red dots denote the PCR products representing variant somatic acquired proviral insertions. M: 100-bp DNA ladder (Taken from Yin, B. et al., BioTechniques 2007. © 2008 BioTechniques. Used by Permission).
Figure 3 Comparison of PCR band patterns amplified from leukemias using SplinkBlunt-PCR and STA-PCR protocols. B112, B68 and B75 are BXH-2 derived leukemia samples. NC: healthy BXH-2 mice tail DNA used as a control. M: 100-bp DNA marker. The white arrows indicate the differential bands. The size of DNA marker in base-pair is noted to the left (Taken from Yin, B. et al., BioTechniques 2007. © 2008 BioTechniques. Used by Permission).
Figure 4 Comparison between SplinkBlunt-PCR and Southern blot assay in detecting differential proviral insertions. The genomic DNA from Ara-C sensitive cell line B117P (lane S) as a passage control, or Ara-C resistant cell line B117H (lane R), was subjected to proviral insertion cloning PCR protocol (A) or Southern blotting analysis (B). Arrows indicate the differential insertion (Taken from Yin, B. et al., BioTechniques 2007. © 2008 BioTechniques. Used by Permission).