Chapter 3 Enzyme manipulation of DNA and RNA
To measure incorporation of radioactivity (to see if the probe is good or not for hybridization) Acid precipitation method: - Add sonicated salmon sperm DNA and cold acid (HCl) solution, - Collect the precipitate on a glass microfiber filter (Whatman GF/A), - Measure radioactivity
To separate radio-labelled probe from dntps Spin column method (Fig. 3.2.1), Resin (Sephadex G-50, Bio-Gel P-60) Silanized glass wool Dye (phenol red)
Labeling RNA by run-off in vitro transcription (unit 2.13) - Linealized DNA by restriction digestion immediately downstream of the fragment to be labeled - Mix: DNA, SP6 (T7, T3 etc) RNA polymerse, A, C, G, and [α-32p]u, RNase inhibitor, and incubate RNase free DNase I incubation - Stop reaction by EDTA, can be stored at -20 0 C for 2 days before use. (During hybridization, use low-stringency wash firstly, Then, RNase A and RNase T1 to remove unhybridized probe, Finally moderate- and high-stringency washes.)
Labeling DNA by nick translation - Mix: DNA fragment da, dc, dg, α-32p]du, DNase I, E. coli DNA polymerase I, and incubate - Stop reaction by EDTA (and trna) - Phenol extraction - Spin column
Labeling DNA by random priming -Mix DNA fragment and random hexanucleotides, boil and ice - Add da, dc, dg, [α-32p]du, Klenow fragment, and incubate - Stop reaction by EDTA (and trna) - Phenol extraction - Spin column
Labeling 3 -end of dsdna with 5 overhang (fill in) Repair 3 or 5 overhangs to become blunt ends all by Klenow fragment
Restriction digestion Complete digestion Partial digestion: by reduction enzyme concentration or digestion time, or combination. (DNA treated with DNA methylase will resistant to digestion of the corresponding RE.)
Synthesis of homopolymer tails (tailing) or biotin-11-dutp labeling by Terminal (deoxynucleotidyl) transferase - template independent - tailing or biotin-labeling to 3 -end of dsdna or ssdna - best with ssdna and dsdna with 3 - overgangs)
Removing of 5 -protruding ends of dsdna (for further tailing by terminal transferase) by Lambda exonuclease (λexo)
Synthesis of cdna by Reverse transcriptase (using either oligo (dt) primers, or random primers) - RT can degrade the RNA in an RNA::DNA hybrid or RNA can be destroyed by NaOH
Dephosphorylation of 5 -end of DNA, RNA, dntps, and NTPs by CIP (calf intestine phosphatase) or BAP (bacterial alkaline phosphatase) - to avoid self-ligation of vector DNA
Phosphorylation of oligonucleotides, ssdna, or dsdna with 5 -OH ends by T4 polynucleotide kinase (transfer γ phosphate of ATP to 5 -OH of DNA, RNA) - for ligation of linkers best with 5 protruding ends, blunt ends are OK. - for RE mapping, ds DNA first dephosphorylated by CIP, then labeled by T4 polynucleotide kinase using [γ-32p]atp, then partial digestion of the labeled DNA.
Exonuclease Exo VII: work on 3 and 5 end of ssdna, processive λexo: work on 5 overhangs of dsdna, processive, work on 5-P T4 gene 6 exonuclease: work on 5 overhangs of dsdna, non- processive, work on 5-P and 5-OH Exo VIII: work on 3 -OH of dsdna, non-processive
Endonclease Bal 31: degrade ssdna, rrna, trna, ss region of ccc, 5 end and 3 end of linear dsdna (controlled shortening of dsdna) S1, mung bean nuclease: degrade ss DNA, ss region of ccc Micrococcal nuclease: degrade DNA and RNA DNase I (RNase-free DNase I): degrade ds DNA, nicks dsdna in the presence of Mg +2 ; generates ds breaks in the presence of Mn +2
Ribonuclease RNase A (DNase-free RNase A): degrade RNA after C and U (inhibited by RNase inhibitor: eg., RNasin from Promega) RNase H: degrade RNA in RNA:DNA duplex. RNase T1: degrade RNA after G
Ligase T4 DNA ligase: ligation between 5 -P and 3 -OH in duplex DNA T4 RNA ligase: ligation of 5 -P ssdna or RNA to 3 -OH of ssdna or RNA.
Sub-cloning - RE, CIP. Klenow, linker, T4 DNA ligase - can be done in low-gelling gel slices
Construction of recombinant DNA molecules by PCR Fig. 3.14.1 Fig. 3.14.2 Fig. 3.14.3
Detection by non-isotopic probes Non-isotopic probes: labeled with biotin or digoxigenin, stable for 2 years.
I. Biotin-labeled probe and detection Biotin binds to streptavidin (4 x 13 kd) tightly.
Biotin probes Nick translation reaction Biotin-11-dUTP is substituted for dttp no phenol extraction (biotinylated probe will go to interphase or phenol layer) Random priming Biotinylated random octamers (NEB, Millipore) Biotin-11-dUTP is substituted for dttp
For QC of biotinylated probes, colorimetric method ( color α biotin/ kb) Dot blot biotinylated standard DNA (Life Technologies) test probe Uncharged nylon membrane Hybridization Streptavidin-alkaline phosphatase (AP) conjugate Nitriblue tetrazolium (NBT) 5-bromo-4 chloro-3-indoyl phosphate (BCIP)
Detection by biotinylated probes Chemiluminescent method (can detect 1 copy of gene in 1μg human genomic DNA) Uncharged nylon membrane blot with DNA or RNA Biotinylated probe Strepavidin Biotinylataed AP Chemiluminescent substrates (eg. Western lightning chemiluminescence Rreagent plus, PekinElmer Life Science)
II. Digoxigenin -labeled probe and detection (Genius kit, BM) Digoxigenin Labeling nick translation or random priming, replacing dttp by digoxigenin-11-dutp/dttp QC by digoxigenin-labelled stand DNA (part of kit) Detection color or chemiluminescent method using AP conjugated anti-digoxigenin antibody.