Direct cDNA screening by large fragment genomic DNA cloning
Direct cDNA screening by large fragment genomic DNA cloning
This protocol uses a 500kb contiguous sequence of human genomic DNA cloned in a BAC vector to directly screen for cDNAs complementary to it, but the method can also be applied to genomic target DNAs of any size.This experiment was derived from the Guide to Molecular Cloning Experiments (3rd edition), by Peitang Huang.
Operation method
Direct cDNA screening by large fragment genomic DNA cloning
Materials and Instruments
Amplification buffer DNA polymerase DNaseI restriction endonuclease ATP streptavidin-magnetic bead binding buffer T4DNA ligase heat-stabilized DNA polymerase agarose gel BACDNA barbed panning buffer Hybridization solution Flat-end cDNA Cot1 genomic DNA cytosol poly(A)+RNA dNTP solution DNA molecular quality markers oligonucleotides Positive control DNA marker compounds Move makings For more product details, please visit Aladdin Scientific website.
Autosampling tips Geiger or Cherenkov counters Heaters Hybridization ovens Oscillating water baths Magnetic separation devices Microcentrifuge tubes Autosamplers SephadexG-50 columns Paramagnetic beads Thermal cyclers Water baths
Buffers & Solutions
10x Amplification Buffer
Buffer containing 0.01% (m/V) gelatin
ATP (10 mmol/L)
2X hybridization solution
1.5mol/L NaCl
40 mmol/L sodium phosphate buffer (pH 7.2)
10 mmol/L EDTA (pH 6.0)
10xDehardt's liquid
0.2% SDS
NaOH (0.lmol/L)
1X Barrier Panning Buffer
500 mmol/L Tris-HCl (pH 7.5)
100 mmol/LMgCl2
50 mmol/LDTT
SDS (10%)
20XSSC
Streptavidin-Magnetic Bead Binding Buffer
10 mmol/LTris-HCl(pH7.5)
1 mmol/LEDTA(pH8.0)
1 mol/LNaCl
Tris-HCl(lmol/L,pH7.5)
Enzyme and buffer
DNA polymerase/DNaseI (5 units/ul; Boehringer Mannheim)
Restriction endonuclease
T4DNA ligase
Heat-stabilized DNA polymerase
Gel
Agarose gel (1%), 0.5XTBE preparation
See steps 18.19, 24 and 25
Nucleic acids and oligonucleotides
BACDNA containing a region of target genomic DNA
Purification methods are described in Chapter 4, Scheme 8 or 9.
Flat-ended cDNA prepared by random initiation
See Scheme 1, Phase 1.
Cot1 Genomic DNA
Cytoplasmic poly(A)+RNA, purified from subject tissue or cell line.
See Protocol 1, Phase 1.
DNA Molecular Quality Markers
dNTP solution, 0.4 mmol/L (pH 8.0) of each dNTP concentration, for notch pan labeling experiments
dNTP solution, 2.5 mmol/L (pH 8.0) of each dNTP concentration for PCR amplification
Oligonucleotides [10 mmol/L, TE (pH 8.0) preparation]
Positive control DNA
Positive control DNA must be part of a gene or expressed sequence tag (EST) known to be present in the initial genomic DNA. If there is no known gene, add a single copy of control DNA to the genomic DNA (in a 1:1 ratio) prior to labeling, and add control DNA to the cDNA sample when sniffing, so that the molecular ratio of control DNA to cDNA is 1:106. Label the DNA samples with one of the protocols given in Chapter 9; the choice of labeling protocol will depend on the purpose of the experiment (see the Introduction to Chapter 9 for a discussion of the choices). The choice of labeling scheme depends on the purpose of the experiment (see the Introduction to Chapter 9 for a discussion of the choices).
Labeling Compounds
[ a-32P ]dCTP (3000Ci/mmol)
Biotin16-dUTP (0.4 mmol)
Equipment
Autosampling tip with cartridge
Geiger or Cherenkov counter
Heating unit with preset temperatures of 14°C and 100°C
Hybridization ovens with rotating shafts or oscillating water baths
Magnetic separation device for the separation of Streptavidin beads
Microcentrifuge tube (0.5 ml thin-walled tube for PCR amplification)
Autosampler with de-tip device
SephadexG-50 column, equilibrated with TE (pH 7.6)
Steptavidin-coated paramagnetic beads
Thermal cycler with pre-programmed amplification protocols
If the thermal cycler does not have a thermal cap, mineral oil or sealing paraffin must be used to minimize evaporation of liquid from the reaction mixture during PCR. The use of sealing paraffin not only prevents evaporation, but also keeps the components (e.g., primer and template) separate until the reaction mixture is heated, which helps to prevent non-specific binding of the primers at the initial stages of the reaction (see "Hot-Start PCR" in the Information section of Chapter 8).
Water bath with a preset temperature of 65°C
Additional reagents
The reagents required for step 1 of this program are listed in Chapter 10, Program 2.
Reagents required for Step 2 of this protocol for random primer initiated double-stranded cDNA synthesis or for amplification of insert fragments from cDNA libraries are listed in Protocol 1, Phases 1 and 2.
Reagents required for labeling in Step 2 of this protocol are listed in Chapter 9, Schemes 3, 5, and 6.
Reagents required for steps 19 and 20 of this protocol are listed in Chapter 6, protocols 8 and 10.
Methods
Preparation of cDNA libraries with junctions
1. According to Scheme 2 in Chapter 10, label the 5' end phosphorylation of Ooligo3 and oligo4 with polyribonucleotide kinase, mix the labeled two complementary oligonucleotides in equal molecular ratios (about 2ug each), denature at 100°C for 10 min, and slowly cool to room temperature. During this process, the two oligonucleotides are complexed to form an articulator. The concentration of the linker is adjusted to 1ug/ml.
2. Prepare at least 2ug of flat-ended double-stranded cDNA from cytoplasmic poly(A) RNA by random initiation (see Scheme l).
3. Configure the following ligation system in a sterile 0.5 ml microcentrifuge tube:
Double-stranded flat-ended cDNA (step 2) 2ug
Oligonucleotide Amplification Kit Mix 3ul
(1ug/ul from step 1)
10XT4DNA Ligase Buffer 3ul
10 mmol/LATP 3ul
T4DNA Ligase (1 unit/ul) 3ul
Add H2O to 30ul
Incubate at 14°C for 16 h. Inactivate T4DNA ligase at 65°C for 10 min.
If ATP has been added to the 10x ligation buffer component, a larger volume of vector or exogenous DNA may be added to the reaction mixture; ATP is not required when using commercial ATP-containing ligation buffers.
4. Extract the ligation product with phenol/chloroform, purify the product by centrifugal column chromatography using SephadexG-50 and precipitate the DNA using the conventional ethanol method. dry the precipitate and redissolve it in 10ul of TE (pH 7.6).
Biotin labeling of genomic DNA clones
5. Introduce biotinylated bases into the BAC genomic DNA clone using notch panning. Configure a sterile 0.5 ml microcentrifuge tube with the following nick-panning reaction system (label each BAC genomic DNA separately).
Purified BAC DNA (0.1 mg/ml) 1ul
Biotin-16-dUTP (0.04 mmol/L) 1ul
10x Barrier Panning Buffer 2ul
dNTP solution for notch panning (0.4 mmol/L) 1ul
[a-32P]dCTP (3000Ci/mmol) 1ul
DNA polymerase/DNaseI (5 units/ul) 1ul
Add H2O to 20ul
React at 4°C for 2 h.
6. The radioactively and biotinically labeled product by notch shift is purified by chromatography on a Sephadex G-50 centrifugal column and the DNA is precipitated by the usual ethanol method. the precipitate is redissolved in 10ul of TE and stored at -20°C. The product is then purified by chromatography on a Sephadex G-50 centrifugal column.
Preparation of Streptavidin magnetic beads
7. In a sterile 1.5 ml mini-centrifuge tube, wash 3 mg (300ul) of magnetic beads three times with 500ul of Streptavidin-Bead Binding Buffer. After each wash, the beads are removed from the binding buffer using a magnetic separation device. After washing, the beads were resuspended in Streptavidin-Magnetic Bead Binding Buffer at a concentration of 10 mg/ml.
8. For each labeling reaction, a small amount of product (step 6) is taken and tested for its ability to bind the streptavidin-coated magnetic beads.
Configure a sterile 0.5 ml microcentrifuge tube with the following binding system
washed streptavidin-coated magnetic beads (step 7) 20ul
Labeled Genomic DNA Continuous Fragment (Step 6) 1ul
Streptavidin-bead binding buffer 29ul
~undefined Mix equal amounts of each BAC DNA labeled separately on the stacked contiguous cluster, the concentration of the mixture is 10ng/ul.
Incubate at room temperature for 15 min, mixing gently from time to time in between. Separate the beads with a magnetic separation device and transfer the supernatant to a sterile 0.5 ml microcentrifuge tube. Determine the activity of the beads and the supernatant with a Geiger or Cherenkov counter. If the ratio of bound to free isotopes is greater than 8:1, direct screening is performed.
If the ratio of bound isotope to free isotope is less than 8:1, it may indicate that the DNA in step 5 is not suitable for labeling. The BACDNA can be purified by repeated extraction with phenol/chloroform and chromatography on a SephadexG-50 centrifugal column prior to labeling.
Direct Screening (First Round of Screening)
9. Block or "repeat suppress" the cDNA library (from the repeat sequence in step 4) with C0tlDNA as follows:
a. Configure the following replication reaction system in a sterile 0.5 ml microcentrifuge tube:
cDNA with junction (step 4) 5ul (1ug)
Human genomic C0tlDNA 5ul (1ug)
b. Add low gravity mineral oil (~50ul) on top of the reaction mixture to prevent evaporation and denature for 10 min at 100°C. Cool the reaction system to 65°C, add 10ul of 2X hybridization solution under the mineral pool. Mix well and incubate for 4 h at 65°C.
If a YAC genomic DNA target is used, the DNA may be contaminated with host (yeast) ribosomal sequences. Block these sequences with at least 100ng of rRNA-derived cDNA (see troubleshooting at the end of this protocol).
10. When hybridization of the cDNA library to C0tlDNA is complete, perform the first round of direct screening. Take 5ul (50ng) of biotin-labeled BAC DNA from step 6 and add it to a new 0.5 ml microcentrifuge tube, covering the top of the reaction solution with a drop of mineral oil (~50ul) to prevent evaporation. denature the BAC DNA at 100°C for 10 min and then cool the reaction system to 65°C.
11. Configure the following replication reaction system in a sterile 0.5 ml microcentrifuge tube:
Biotin intercalated BAC DNA (from step 10) 5ul (50ng)
Enclosed cDNA (from step 9, 1ug 20ul)
cDNA plus 1ug C0tlDNA )
2x hybridization solution 5ul
Mix well and incubate for more than 54 h at 65°C in a rotary hybridization oven or in an oscillating water bath.
12. For capture and washing of genomic DNA and cDNA hybrids, add the following reagents to a sterile 1.5 ml microcentrifuge tube:
100ul of washed streptavidin-coated magnetic beads
Recombination reaction mixture (from step 11) 30ul
Streptavidin-bead binding buffer 100ul
Incubate at room temperature for 15 min with occasional gentle mixing. Attach the beads with a magnetic detachment device and remove the top.
Wash the beads twice with 1 ml of 1xSSC/0.1% SDS at room temperature for 15 min each time; then wash the beads three times with 1 ml of 0.1xSSC/0.1% SDS at 65°C for 15 min each time, and transfer the beads to a new microcentrifuge tube after the last wash.
13. Elute the cDNA hybridized to the beads in step 12:
a. Add 100ul of 0.1mol/L NaOH and allow to stand at room temperature for 10 min. b. Add 0.1mol/L NaOH to the beads.
b. Add 100 ml/L Tris-HCl (pH 7.5).
c. Desalt the reaction mixture by centrifugal column chromatography on a Sephadex G-50 column (see Appendix 8).
Amplification of cDNA from the first round of screening
14. From the eluted 200ul of cDNA (step 13), three portions (1ul, 5ul and 10ul) are added to a sterile 0.5 ml PCR tube.
15. Add the following reagents to each tube:
Primer oligo3 (10 mmol/L) 5ul
10XPCR buffer 2.5ul
dNTP solution (2.5 mmol/L each) for PCR 2.5ul
Taq DNA polymerase (Perkin Elmer, 5 unit ul) 0.2ul
Add water to 25ul
Set up two control experiments simultaneously in the above configuration. The negative control tube is filled with all the above reagents, but the cDNA template is omitted. For the second control, add 10ng of initial cDNA (step 4) as template.
16. If the thermal cycler does not have a thermal lid, cover the top of the reaction mixture with a drop (~50ul) of low gravity mineral oil to prevent evaporation. Alternatively, add paraffin beads to apply a hot-start PCR. place the PCR tube on the thermal cycler.
17. Perform the amplification reaction with the denaturation, denaturation and extension parameters listed in the table below. 
18. 10% of the PCR product from each tube is analyzed by electrophoresis on a 1% agarose gel in 0.5 XTBE electrophoresis buffer, with the appropriate size DNA molecular mass standard. The gel is stained with ethidium bromide or SYBR GOLD to visualize the DNA, to estimate the concentration of cDNA amplification products, and to determine the concentration of template cDNA that should be added to maximize amplification products.
The product of a successful reaction will be seen as a diffuse cDNA amplification fragment, possibly with some clear bands.
19. Take an equal amount of amplification product from each tube (approximately 0.5ug per lane) and add it to the second 1% agarose gel, again adding the appropriate size DNA molecular mass standard. Simultaneously sample 0.5 of the cDNA prepared by randomization in Step 4.
20. Transfer the separated DNA to a membrane and hybridize to the radiolabeled positive control cDNA by Southrn blotting (Scheme 8, Chapter 6).
The hybridization signal produced by the screened cDNA should be approximately 1000 times stronger than the hybridization signal produced by the randomly initiated cDNA (Step 4).
21.- Once enrichment of the positive control has been confirmed, amplify a large volume to prepare at least 1.5ug of the screened cDNA.
The reaction product is extracted with phenol/chloroform and the DNA is precipitated using the standard ethanol method. The precipitate is dried and redissolved in 7.5ul of TE (200ug/ml).
Direct cDNA screening (second round of screening)
Follow the same conditions as in the first round of screening, using 1ug of cDNA from the first round of screening and 50ng of genomic target DNA (in this case, 500kb of full length).
22. Sequence closure of repetitive sequences in the cDNA screened in the first round as described in step 9 (use 1ug of the first round screening product and retain 0.5ug for subsequent analysis).
23. Perform a second round of screening as described in steps 9 through 13.
24. Analyze the amplified products from the second screen by 1% agarose gel electrophoresis as described in step 18, sampling 0.5ug of the retained first screen cDNA product in a separate lane, along with 0.5 of the initial cDNA.
25. Perform Southern blot analysis with radiolabeled reporter probe as in step 20.
Typically, the results of this analysis will show that the abundance of the reporter probe is greatly increased from the initial cDNA to the first screen cDNA, and that the abundance of the reporter probe is relatively moderately increased (approximately 10-fold) from the first screen cDNA to the second screen cDNA.
26.- Once the enrichment of the positive control has been confirmed, clone the screened results into a suitable vector. The restriction sites on the amplification kits facilitate the cloning of cDNAs from the second round of screening into phage or plasmid vectors.
A useful alternative is to use one of the now commercialized ligation-independent cloning systems (e.g., Life Technologies UDG System or CLONEAMP pAMP Vector System). If one of these systems is used, the 5' oligo3 primer with a tinted stretch modification is used (see manufacturer for details). For an in-depth discussion of this strategy, see the information section "Linkage-independent cloning". 