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Gene (遺伝子)






Mount Usu / Sarobetsu post-mined peatland
From left: Crater basin in 1986 and 2006. Cottongrass / Daylily

Gene (遺伝子)

sequence of nucleotides in DNA or RNA that encodes the synthesis of a gene product, either RNA or protein

Nucleic acids (核酸)


1869 Miescher, Friedrich (1844-1895): nuclein extracted from pus
1871 Hoppe-Seyler EFI (1825-1895) & Miescher F

nuclein extracted from red blood cell, yeast, yolk, salmon soft roe, etc

→ they did not detect the function

Structure

a) nucleotide = [base]–[sugar]–[phosphate acid]

N-base___ nucleoside
thymine___thymidine
adenine___adenosine
guanine___guanosine
uracil_____uridine
cytosine__ cytodine

cf. nucleoside: [base]–[sugar] → triphosphate deoxynucleoside, distributed in nuclei

A-dR: deoxyadenosine_A-R: adenosine___dATP
G-dR: deoxyguanidine_G-R: guanosine___dGTP
C-dR: deoxycytidine___C-R: cytidine_____dCTP: phospholipid
__________________________________ synthesis
T-dR: deoxythymidine__×______________dGTP: protein synthesis
×__________________U-R: uridine

b) pentose (五炭糖)

DNA (= deoxyribose): H bounded at C2' ↔ RNA (= ribose): OH bounded at C2'
na na
________________Ribose_____________Deoxyribose
sugar conformation
2'-exo 3'-end_______________2'-endo 3'-exo
na na

c) base
                    relative   base  lg (μm)       MW
                    volume     pair

DNA virus    SV40             4,000      1.6       3.1·106
             λ phage        53,000     16        31·106
    bacteria E. coli      3,400,000 12,000     2,320·106
RNA          m-RNA     5%    random               -4·105
             t-RNA    15%     70-80                2.5·104
             r-RNA    80%       120                3.6·104
                              1,700                0.55·106
                              3,700                1.2·106

Purine group (プリン族): purine nuclei (consisting of five-membered and six-membered rings) = adenine (A) and guanine (G) → C1 on the pentose replaced from H on N9 when nucleic acid is formed

baseG, baseA

Pyrimidine group (ピリミジン族): Pyrimidine nuculei (consisting of six-membered rings) = Cytosine (C), Uracil (U), Thymine (T) → C1 on the pentose replaced from H on N3 when nucleic acid is formed

baseC, baseU, baseT

Abnormal base (異常塩基)
base R = base Y
base
2'O-methyl nucleoside [2'Om (A, C, G, or U)]

7-Methyl guanosine (7mG)___Inosine (I)

base
N2-Dimethyl guanosine (2dmG)

N6-ΔIsopentenyl 2 methylthioadenosine (2ms6iA)

base
N6-ΔIsopentenyl adenosine
base
Dihydrouridine (D) Pseudouridine (ψ)_4-Thiouridine (4SU)
索引

Deoxyribonucleic acid, DNA (DNA)


Extraction of DNA from DNA-protein complex

DNA-protein complex (including RNA)
  1. cell disruption
  2. celll fractination – nuclei, organelle, chromatin
  3. protein-denature (DNA, protein – disordinate)
    1. pronase: use SDS to remove possibility that RNase is working
    2. detergent: denature protein (Ex. sarkosyl SDS)
    3. salt concentration = 1-2 M NaCl
    4. phenol, chloroform
Buffer:

0.1M Tris-HCl (pH 7.5-8.0), 0.1-0.2 M NaCl, 1-5 mM EDTA, homogenized with 1-2% SDS

Structure of DNA (DNAの構造)


Circle DNA (環状DNA)
i) Open circle DNA (o.c. DNA)
ii) Closed circular (c.c. DNA) (= coralently closed circular DNA, superhelix, supertwist, super coil)

relaxed circlar DNA____super coil
_______DNA
Twisting returns when one part is cut (constraint: distortion occuring in the DNA chain due to forcibly approaching)

DNA replication (DNA複製)


DNA polymerase (DNA合成酵素)

1956 Kornberg et al.: discovered from E.coli in the experiment of in vitro DNA replicaiton

dXTP = uptake into primer DNA
DNA
60': purified = Kornberg enzymes (= DNA polymerase I, poly I)

MW 10900, single-strand peptide, one SS-bond
→ function = mending enzyme for DNA

i) high exonuclease activity

response to both 5'-end and 3'-end
fragments dichotomize by papain has activity → all of them form 5'-mononucleotide

ii) pyrophosphorylase activity present

5' → 3': transfer the next nucleotide to 3'-OH, indicator free 3'-OH
→ these are mending enzymes

Necessity
  1. template (鋳型) – synthesized DNA is complementary to the template
  2. primer (出発点)
1963 Richardson, Schildkraut, Kornberg: E. coli

DNA
____________Exonuclease III______DNA polymerase I
______________________partly single______repair
cf. exonuclease DNAendonuclease
__cut from outside______________cut from inside

1969 Cairns & DeLucia: pointed out issues on poly I as DNA synthetase

discovered defective mutant of DNA polymerase I(A) from 3478 strains → DNA polymerase I is not essential for DNA synthesis

1969 Cairns et al. +
1970 Kornberg & Gefter +
1970 Moses & Richardson +
1970 Knippers

DNA polymerase II

1970 Kornberg T

DNA polymerase III: RNA as primer, DNA as template, 5' → 3'

1970 Temin, Baltimore

discovered reverse transcriptase = RNA-dependent DNA polymerase

1971 Wang: E. coli W-protein – DNA binding protein

dna genge: engaged in gene replication
dna B, dna C = primer RNA
dna E (pol C) = DNA polymerase III
dna G = primer
dna Z = poly III &gamm;a subunit
pol A, pol B
lig (ligase)

DNA polymerase
E. coli: poly I(109Kd), II(120Kd), III(140Kd) & III*
Mammals: poly I(α), II(β), III(γ)
DNA polymerase I
DNA dependent
Okazaki fragment lacks the primer
RNA polymerase does not need the primer
→ A hypothesis proposed by Okazaki is that: firstly RNA is formed and then DNA is formed by removing RNA later

DNA

DNA polymerase II
DNA polymerase III* |α 140| Poly III (dna E, pol C)
                    |ε 25 |
                    |θ 10 |
                    |J 83
                    |γ 32   dna Z      |factor II
                    |δ 32   factor III |
                    |β 40   factor I – copol III*
Three DNA polymerases, α, β and γ, in eukaryotes
α: 12-13 MW

high ion intensity > 25 mM
N-ethylmaleimide (NEM) – restraint the activation by attaching SH (sensitive)

β: 3-5 × 104 MW (low molecular)

utilizing oligo12-18, poly(rA)
SH-group, nonsensitive to NEM (→ distinguished from α)
stimulate (activity↑) under high ion intensity
not related to cell cycle? – chromatin bound polymerase

γ: > 1 × 105 MW

favor oligo
NEM sensitive
like-mitochondrial DNA

Plant DNA polymerase
1974 Mory, Chew & Sarid: DNA polymerase extracted from wheat
1978 Stevens & Bryant

mashing up pea seedlings &larr ; collecting supernatant after centrifugation → ammonium sulfate concentration gradient → extracting animal-like α, β DNA plymerases

1979 Horemann & Follmann

DNA synthesis by Vicia fava
Measuring the activity of ribonucleoside-5'-diphosphate reductase → DNA polymerase governs the supply of substrates

DNA repair (DNA修復)

1) Gilbert

neck____Enzyme (= ligase joining enzyme)
-------------____-------------
------ ------______-------------

2) 1962 Setlow & Setlow
3) 1967 Gilbert

replication
T4 phase gene 30 = ligase → However, the mutant (ts B50) does not synthesize normal ligase under high temperature

Trigger of DNA synthesis (DNA合生開始)
Semi-conservative DNA replication (半保存的DNA複製)
1960 Matsuoka: Chlamydomonus (eukaryotes), 1961 Simon: Hela cell

Table. Structure of polynucleotide double helices

1967-1970 Inman

λ-DNA: eye shape = double strand
______________start at gene 0 of which length is 3 μm
synthesis
synthesis

1962 Kleinshmidt: success in first observation of DNA by using an EM

NA, cytokinine c–basic + 2M ammonium acetate
sub-mutant → genetic code
UAA, UGA, UAG: nonsense code
TS mutant → used for DNA synthesis

1966 Edgar RS: T4φ: approximetely 10 are concerned with the replication synthesis

30, 32, 41, 42, 43, 45, 46, 47, 56, 62
30: DNA ligase (→ bound with DNA)
32: DNA binding protein
43: DNA polymerase

1967 Albert

Replication (複製)

1) Eukaryote

nucleolus-DNA, r-RNA genes → replications [special example]
amplification (増幅): specfice gens increasing reversively at a life cycle in higher organisms; R-RNA genes increasing from somatic cells

2) Chloroplast DNA (Kolodner & Tewari 1975)

1st: Caris type (θ) → 2nd: Rolling type

2’) Mitochondria DNA (Robberson, Kasamatsu & Vinograd 1972)

a___________b_____________c
replication"
starting the replication to the inverse direction just after the replication of a repeating this process

Sites for DNA replication (DNA複製の場所)
Hypothesis: eukaryotic body cells (2n → DNA replication starting for nuclear membrane
1965 Sparvoli, Gay & Kaufmann

Tradescantia - prochromosome

1965 DuPraw

chromatin fiber ≈ 230Å

1970 Comings & Okada

at metaphase on Chinese hamster, etc. → chromatin bounded with nuclear membreane

1973 Huberman, Tsai & Deich
1976 Sparvoli, Galli, Mossa, & Paris

Haplopappus gracilis, n = 2
Condensed chromatin → synthesis occurring at the late S stage
Diffused chromain → synthesis occurring at the early S stage

1974 Berezney & Coffy, 1976 Kelly & Riley, 1978 Miller, Huang & Pogo
Table. Structure of polynucleotide double helices. *: These have parallel strands. All others have anti-parallel strands.
SampleRelative humidity (%)Residues per turnTranslation per residue (Å)Angle between base planes and helix axisDihedral angle between base planesSugar conformation
DNA-A, Na+75112.5570o16°C3'-endo
DNA-B, Na+95103.46
DNA-B, Li+66103.3788°C2'-endo
DNA-C, Li+669.33.3284°10°C2'-endo
DNA-RNA hybrid, Na+75112.6270°
Yeast RNA fragments7510 or 112.9 or 2.64
Reovirus α or β forms7510 or 113.0 or 2.73
poly rA·rU10 or 113.1
poly rI·rC123.0
poly rCH+·rC*123.11
poly rAH+·rAH+*83.8

Ribonucleic acid, RNA (RNA)


= A + G + C + U
  • mRNA (messenger RNA)
  • rRNA (ribosomal RNA)
  • tRNA (transfer RNA)

16S ribosomal RNA (16S rRNA)

Prokaryota - 16S rRNA gene
used in reconstructing phylogenies, because of the slow evolution rate of this region of the gene
16S ribosomal databases
EzBioCloud (former EzTaxon)
Ribosomal Database Project (RDP)
SILVA
GreenGenes
Gut bacteria: global rumen census datasets (Henderson et al. 2019)

Gene expression (遺伝子発現)


Reversed genetics (改変遺伝学)

gene engineering (遺伝子工学) = gene manipulation (遺伝子操作)

isolation of gene → assay of gene structure → restriction map (制限酵素地図)
reversed genetics

designed alteration of the gene

gene structure can be altered freely by the researchers who use reversed genetics

fraction test of the mutant (mutated) genes (J Genetics 57: 581-598, 1982)
reversed genetics
→ investigate the mechanisms of transcription =
I. in vitro (cell free) system
II. cultured cells: injection, Ca phosphate gel
III. fertilized egg

I. identification of signal DNA sequence
finding out the components responsible for the function
→ fractionation of extract → purification → rreconstitution → addition of regulatory components
→ establishment of basic transcription system: the amount of transcription can be adjusted by using different developmental stages → clarifying the components of regulation
II. types of cells
Basic transcriptive function
=============== DNA signal: utilized heterogenous cells
Regulatory function
-------------------------- cell that expressed, per se
reversed genetics
____________________________
when cells containing different genes are inserted, endogenous or exogenous genes can be detected
reversed genetics
III. fertilized egg
reversed genetics mouse/Drosophila

→ test of regulated expression → identification of signal DNA sequences

Finding out the component responsible for the regulated function

Molecular and developmental biology (分子発生生物学)


(Suzuki & Brown 1972)

Isolation of sil fibroin mRNA
crude mRNA for globin mRNA (only the way at that time) (Williamson & Paul 1972)
crude mRNA: labeling → hybridization with DNA → -104 genes/haploid: required pure DNA
Table. Bombyx mori: the amount of amino acids is measurable, and the sequence is partially determined
                 Glycine Alanie Serine         Tyrosine  Others

  mol%             45      29      12             5         9
  genetic codon   GGX     GCY   UCZ or AG[U/G]  UA[U/C]

G/G content = high
G: (30 + α mol%) + (9.3 + α) ≈ 40 + α
C: (9.3 + α) + (2 + α) + (1) + (1) ≈ 12 + α
G + C = 57–60% ⇒ such mRNA should be present

The amino acid sequence of fibroin clarified at that time

60% (w/w):_____(Gly-Ala-Gly-Ala-Gly-[Ser-Gly-(Ala-Gly)2]8-
_________________Ser-Gly-Ala-Ala-Tyr)n = 58 A.A.
40%:______5.8% Gly-Ala-Gly-Ala-Gly-Ala-Gly-Try
_______________------------------------- alliance
__________5.6%
__________4.5%
⇒ consider codon
GGX-UCZ or AGU/C-GCY-GGX-GCY-GGX

in Rnase T1 (cut 3'-side of G)

Finger print: consider UCZ case (many Z = A)
    Residue      G    XG   CYG XUCZG  Total
    Nucleotide   3     4     6     5     18

*: if X or Y is G, the bond is cut → X and Y are not G

Use DEAE-Sephadex
Profile made by UD260, 32P

Advantages of silk fibroin = 16000 nucleotides, 4.4% of total DNA
finger print

Detection of fibroin genes
1972 Suzuki, Gage & Brown

Ex. fibroin mRNA 90%: 1 copy/haploid → 90
___contaminant 10%: 100 copy/haploid → 1000

not matched with the fact

finger print
total genome G + C = 39%
fibroin mRNA (gene) ~60%

DNA in rear-part silk gland cell ........ 1-3
DNA in middle-part silk gland cell .... 1-3
DNA in the whole body .................... 1-3
⇒ number of copies in the whole body is equivalent

fibroin gene = 0.002 × 2 (%) of total genome
[Conclusion] (Gage & Manning 1976): 1 copy/haploid
Cf. analbumin gene (Sullivan et al. 1973), globin gene (Harrison et al. 1974)

Exeption: Drosophila chorisin(s) in egg (Spradling & Mahowold 1980)

4 kinds out of ca. 50-70% kinds
amplification: can not be observed in general genes
Cf. Prokaryotes – Sarmonella phase variation (transition)

Structural analysis of the genes
Intron discovery (イントロン) (Cold Spring Harbor Symposium, Quantitative Biology 1977)
Intron was firstly confirmed by using adeno-virus 2 DNA (chromatin)
           ├──→ 
       ──┼───────── early gene
    5' ──┼───────── late gene (functional)
           │←──
DNA-RNA hybridization: EM level

Mature mRNA
intron intron

========== "intron" sequence

all sequences are transcribed at first, and then unnecessary portions are discarded

complementary DNA obtained from the mRNA of mouse globrin (Tilghman et al. 1977)

R-loop method (Thomas et al. 1976)
a)__intron
            5'                         3'
    ────┼────────────┼───
    ────┼────────────┼───
            └──↑─────────┘
            1 kb  1kb  intron   15 kb   ⇒ mRNA = 16 kb

the lengths of fibloin fibers are different between the strains - the lengths of DNA are proportionally changed

b) intron existing like this in nature

intron

── exon, ━━ itron

⇒ 1 globrin gene(Tsujimoto & Suzuki 1979ab)
Figure (a) shows that mRNA is artificially cut at the point (↑)

⇒ (ProNAS 74: 4406, 1980)

intron

1981 Flavell RA: Rabitt globulin cDNA (complementary DNA) clone

========================⇒ mRNA
____↓ reverse transcription
========================⇒
←------------------------------------------
_____________
⇐========================
------------------------------------------→

blotting
Southern blotting
Total DNA
↓ restriction enzyme A
DNA digest

electrophoresis
↓ EtBr
blotting blotting 0 × ssc
20 × 0.15 M NaCl,
0.015 M sodium citrate


radioautograph after single strand made by alkaline treatment

⇒ two fragments obtained by using enzyme A
blotting

Jeffreys & Flavell 1977
blotting
1978 Tilghman et al. blotting

blotting
hybridization with cDNA

friend cells 8 l
↓ DM 80
globin ↑







R-loop
blotting

1981 Busslinger, Moschonas & Flanell

β+ thalassemia (地中海性貧血症) – produced globrin somehow
blotting

×: mutation having the genes in the outside

mutation [normal = G, β+ = A] at 21 bp
slow transcription rate at beginning ← splicing can not be made to mature mRNA

blotting

(Kole & Weissman 1982)

in vivo splicing
Hela cell

_________________Nomal splicing__Abnormal splicing
1 Ca/Ph gel β+gene_____100%____________0%
2 Ca/Ph gel β+gene______10%___________90%

in vitro splicing: confirming protein synthesized in vitro (Manley et al. 1980)

Hela cell whole extract
succeed in transcription of most cellular components extracted by 0.42 M (NH4)2SO4 are transcribed in vitro
protein = 1-10 mg ⇔ protein obtained by in vitro splicing = 20 mg/ml (high concentration)
240 μm Hela extract
10 μCi 32P-UTP, CTP, GTP, ATP
100-200 μg/ml DNA (human β globin DNA)
400 μl reaction mixture

Trancation assay
blotting

Synthesizing precursor in vitro

        ↓ phenol extraction   ↓ no phenol
        ↓ new + extract       ↓ extract
        ↓ incubation          ↓ incubation
        ↓                     ↓
        RNA degraded            Stable

probably proteins connected to the synthesized RNA - (possibly) due to splicing
blotting
probably transcription starts from +1

1979 Tsuda, Ohsima & Suzuki

Cell → Total cellular RNA: *32P blotting
mRNA → (reverse transcriptase, RT) copy DNA → cloning (hybridized with natural DNA) ※ shotgun method

Transcription initiation

SI mapping method: method to determine initiation point

actual signal for starting transcription (ex. AGT) = promoter

Hybrids arrest = ex. clone for few numbers of mRNA

Use of in vitro transcription system (生体外翻訳系利用)
RNA polymerase of eukaryotes

Type I gene = rRNA: rDNA = class I gene
Type II gene = general mRNA: genes for mRNAs = class II gene
Type III gene = tRNA, 5S RNA: tDNA, 5S DNA = class III gene

firstly, transcription of type III gene was analyzed

Type I gene: ETS (external transcribed sequence), ITS (internal transcribed sequence) and coding region
Type II gene: modified RNA polymerase II genes

Manley et al. (1980): Hela cell extract
- Promoter on fibroin genes
trancation assay (Tsujimoto et al. 1981) pBR322______________+1_______+514:

  ------------===============▆▆▆▆▆▆▆▆▆▆▆
	                     =========▶
  ----------------===========▆▆▆▆▆▆▆▆▆▆▆
  ---------------------======▆▆▆▆▆▆▆▆▆▆▆
  -------------------------==▆▆▆▆▆▆▆▆▆▆▆

_________________
_________becoming inactive how long the genes are cut
__promoter
*: Class II genes = TATA box (majority): important for transcription

these can be separated by an electrophoresis because the lengths (or masses) of mRNA are different

signal can be activated when the region of -29~+6 is present
promoter
⇒ analyze this sequence - single base is solely important (Hirose, Takeuchi & Suzuki 1982)

Posterior silk gland extract (Tsuda & Suzuki 1981)

Used the method proposed by Manley et al. = 0.4 M (NH4)2SO4
trancation assay: synthesized by 32P-UTP, CTP, GTP and ATP
promoter
promoter
expression level of transcription is two to three times hihger when -860 is contained in silk gland (or serictery) than when not
Used the extraction of Hela cell and stink bug
promoter
promoter
____A and B are the same

Experiment (実習)


Assay by using cultured cells (培養細胞利用検定法)

interferon (human) β gene in plasmid (PSV2-Ecogpt)
↓ CalPh gel
mouse FM3A

2.3 copies of β genes/cell
New castle desease virus = poly(I):poly(c)
    Cells   +                 –       +
          Mouse            (Virus)  Human b

    FM3A   2187             < 6     < 6
    F1      729             < 6     < 6
    F2      729             < 6     162 (6 × 27)
    F3     2187 (729 × 3)  < 6      54 (6 × 9)
    F4      729             < 6      18 (6 × 3)
    F5      729             < 6     162

(Ohno & Taniguchi 1982)

Remkawitz et al. (1982)

Chitin lysozyme gene11
Chitin lysozyme gene + thymidine kinase seequence
↓ (culture: various parts collected from chitin)
chichin oviduct cell (+++)
chichin macrophase (–)
chichin fibroblast (–)
rat fibroblast (–)

1: + steroid hormone →

cells endocytose fluorescent substance to thymidine kinase
→ assay by the gene
→ the substances induced by hormone excite DNA

Δ-161~+15: thimidine kinase gene

Assay system by using fertilized egg
Brinster et al. (1982) experiment
    # of PMK    +K
    molecules

               -cd     +cd
       0       8.5      8.8
      20       8.3     12.2
     200      25.3    270
    2000     190     2400

-1.7 kbp = remaining: 0K, -99 bp leaving: 0K

PCR (polymerase chain reaction, PCR)


DNA marker (DNAマーカー)

RAMP (random amplified microsatellite polymorphism)
SSCP (single strand conformational polymorphism)
PCR-RFLP [CAPS (cleaved amplified polymorphic sequences)]
AFLP (amplified fragment length polymorphism)

Terminal Restriction Fragment Length Polymorphism (T-RFLP) Protocol

Preparing a sample for T-RFLP analysis is a two step process. First, PCR amplify the desired region of DNA using a HEX labeled forward primer and/or a FAM labeled reverse primer. Second, digest the PCR product with one or more restriction enzymes. The details of this process follow.
PCR amplify DNA using labeled primer(s)
Working concentrations of components:
    Component                  Working concentration
    Unlabeled forward primer             10 μM
    Unlabeled reverse primer             10 μM
    HEX labeled forward primer           10 μM
    FAM labeled reverse primer           10 μM
    PerkinElmer PCR Buffer II            10X 
    PerkinElmer MgCl2 Solution           25 mM
    dNTP's                                2 mM (each)
    BSA                                  20 mg/mL
    PerkinElmer AmpliTaq DNA Polymerase   5 U/μL
Volumes (μL) of components per reaction using working concentrations noted above:
                     Reaction
    Component        25 μL   50 μL   75 μL  100 μL
    Forward primer    0.50     1.00     1.50     2.00
    HEX forward pr.*  0.75     1.50     2.25     3.00
    Reverse primer    0.50     1.00     1.50     2.00
    FAM reverse pr.*  0.75     1.50     2.25     3.00
    Buffer            2.50     5.00     7.50    10.00
    MgCl2             3.75     7.50    11.25    15.00
    dNTP's            2.50     5.00     7.50    10.00
    BSA               0.05     0.10     0.15     0.20
    milliQ H2O       13.35    26.70    40.05    53.40
    Taq               0.10     0.20     0.30     0.40
    Template          1.50     3.00     4.50     6.00

* NOTE: Run separate PCR reactions for each labeled primer (i.e., run HEX labeled forward primer with unlabeled reverse primer; run FAM labeled reverse primer with unlabeled forward primer).

Thermal cycler (サーマルサイクラー)

= thermocycler, PCR machine or DNA amplifier
a laboratory apparatus most commonly used to amplify segments of DNA via the PCR
PCR
Using the parameters above, amplify DNA using labeled primer(s)
Check PCR product by agarose gel electrophoresis

- If positive, purify product
- If you have both HEX and FAM labeled product, purify separately and keep them separately until digestion (see below)

Digest the PCR product
Working concentrations of components:
  Component                    Working concentration 
  Labeled PCR product*         250-300 ng (each) per digest
  GibcoBRL REact buffer        10X
  (restriction enzyme specific)
  GibcoBRL restriction enzyme  10 U/μL
  milliQ H2O                   na

* NOTE: If you have both HEX and FAM labeled product, combine 250–300 ng of each labeled product in a single digest (i.e., 250 – 300 ng of HEX labeled product combined with 250–300 ng of FAM labeled product to give a total of 500–600 ng of labeled product in a 20 μL digest)

Volumes (μL) of components per digest using working concentrations noted above:
    Component            Volume (20 μL digest)
    Labeled PCR product  depends on PCR product
                         concentration 
    Buffer               2.00
    Restriction enzyme   1.50
    milliQ H2O           dilute digest to 20 μL
                         (PCR product + H2O = 16.5 μL
                         + buff. + re. enz. = 20 μL)

Digest purified PCR product with preferred restriction enzyme(s)

- If digesting with multiple restriction enzymes, do a separate digest for each restriction enzyme

Digested product is ready for T-RFLP analysis

Protocol provided by Brian Wade at the MSU Center for Microbial Ecology

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