Gii trnh t th h mi

Phm Hng Vn
Trng n V K Thut Phn Tch Gene
Hai
giai on
trong
gii
trnh t
Sanger
ng dng gii trnh t Sanger
1. Gii trnh t gene hay b gen cho cc nghin cu c
lin quan;
2. Pht hin cc t bin gene (lin quan n ung th,
n khng thuc), cc SNP (trong c nhn ha cc
liu php iu tr), cc kiu gene (genotype ca
virus gy bnh);
3. nh danh vi khun hay vi nm da trn gii trnh t
DNA ca gene qui nh RNA ca ribosome (16S ca
vi khun v 28S ca vi nm) c bit l cc tc nhn
kh nh danh hay tht bi nui cy t mu th;
4. Phn tch on xt nghim du vn tay DNA (DNA
finger printing) nhn dng c nhn v mi quan
h c nhn (php y), trong chn on tin sanh,
trong pht hin a dng loi
Pyrosequencing
Nguyn tc pyrosequencing
1. A, hay T, hay C, hay G c lp trnh cho vo ging
phn ng; v mi khi dung dch nucleotide cho vo c
nucleotide c bt cp vo si khun th mt
pyrophosphate (PPi) s c phng thch ra c
enzyme sulfurylase to ra mt ATP, ATP ny s gip h
thng pht quang luciferin-luciferase (luciferin thnh
oxyluciferin pht quang)
2. dATP-S c s dng thay th dATP v dATP-S
khng c hot tnh ca ATP.
3. ATP v cc nucleotide t do cn tha sau mi ln b
sung nucleotide b hu nh apyrase c cho vo
ging sau khi tn hiu pht quang c ghi nhn
nh lng c t bin
ng dng pyrosequencing
Nhiu b thuc th c chp nhn IVD
(In-Vitro Diagnosis) trong pht hin cc t
bin gene ung th lin quan n ch nh v
theo di iu tr ch,
Epigenomic: nh lng cc CpG vi C b
gn methyl (Cytosine methylation) tin
on ung th,
Pht hin t bin gene trong chn on v
sng lc bnh di truyn,
c bit trong vi sinh lm sng: nh danh
cc vi khun.
Ba giai on
ca
gii trnh t
th h mi
Gii trnh t bng tng hp
th h mi vs pyrosequencing
1. Thuc th gii trnh t c thi chy vo chip nano-
well hay vi bn theo chng trnh v sau khi ghi
nhn c tn hiu th thuc th c s c thi chy
ra thuc th mi li c bm vo.
2. Tng hp mch b sung da trn mch khun c th
l ko di u 3 ca mch b sung bng cc
nucleotide (A, T, C hay G) c ghi nhn, hay c
th l ko di u 3 ca mch b sung mi ln 2
base nh s ko di v ni probe da trn si khun
3. Tn hiu tng hp c th l tn hiu pht quang da
trn h thng luciferin-luciferase (454 ca Roche),
tn hiu in do thay i pH (Ion-Tolent hay Ion-
Proton ca ABI), tn hiu hunh quang c nh
du trn cc nucleotide A, T, C hay G (Solexa ca
Illumina), hay cng c th l tn hiu hunh quang
c gn ln probe (solid ca ABI).
Cng ngh Barcode
cho php xc nh ngun gc trnh t c gii
ng dng gii trnh t th h mi
Gii trnh t b gene, thm ch 24h cho b gene
ngi
Li ra nh sng bt c mt trnh t nucleic acid
ca bt c tc nhn g c mt trong mu th ly
t vt ch hay bnh nhn
Cng c nhy cm nht c th pht hin cc
t bin cho d t l t bin hin din trong
mu th l rt thp
Chn on tin sanh pht hin d bi ca thai
nhi bng phn tch DNA ca bo thai hin din
trong mu m da trn u im nhy cao
ca k thut
Gii trnh t
b gene
(Crag Venter)
ng dng gii trnh t th h mi
Gii trnh t b gene, thm ch 24h cho b gene
ngi
Li ra nh sng bt c mt trnh t nucleic acid
ca bt c tc nhn g c mt trong mu th ly
t vt ch hay bnh nhn
Cng c nhy cm nht c th pht hin cc
t bin cho d t l t bin hin din trong
mu th l rt thp
Chn on tin sanh pht hin d bi ca thai
nhi bng phn tch DNA ca bo thai hin din
trong mu m da trn u im nhy cao
ca k thut
ABL1 EGFR GNAS MLH1 RET
AKT1 ERBB2 HNF1A MPL SMAD4
ALK ERBB4 HRAS NOTCH1 SMARCB1
APC FBXW7 IDH1 NPM1 SMO
ATM FGFR1 JAK2 NRAS SRC
BRAF FGFR2 JAK3 PDGFRA STK11
CDH1 FGFR3 KDR PIK3CA TP53
CDKN2A FLT3 KIT PTEN VHL
CSF1R GNA11 KRAS PTPN11
CTNNB1 GNAQ MET RB1
FFPESample
QualityControl
qPCRbased
samplequality
control
Ensure
sampleswillbe
successfulin
assay
Prepare
Amplicons
Prepare
librariesw/
TruSeq
Amplicon
assay
Indexupto96
samples
Builtinsample
normalization
Sequenceon
MiSeq
Simplesample
sheetcreation
withIllumina
Experiment
Manager
Bidirectional
amplicon
sequencing
with2x150bp
reads(2x250
soon)
>75%bases@
>Q30(>99.9%
accuracy)
Automated
analysis
Alignreads
withMiSeq
Reporter
Calllow
frequency
variants(<5%)
Analyzedata
withAmplicon
Viewer
New!
Kt qu gii trnh t b genevirustrn
my MiSeq Illumina
Cc bc tin hnh:
Bo qun mu v bt hot chng virut Rubella
bng dung dch RNA Shield ca hng Zymo
Research M.
Tch chit mu Virut Rubella bng Direct-zol
RNA MiniPrep Kit. (P/N: R2050) ca hng Zymo
Research .
Chun b th vin vi b kit TruSeq RNA LT ca
Illumina.
Chy gii trnh t vi b kt MiSeq 300 cycle V2
trn MiSeq.
Phn tch kt qu s dng phn mm SeqMan v
Blast trn NCBI
Tng thi gian chun b mu v chy gii trnh
t, phn tch kt qu: 3 ngy.
Kt qu
Phn tch k qu bng phn mm SeqMan thu
c ton b genome ca virut Rubella
(9.762bp)
So snh trnh t thu c trn
ngn hng gene NCBI
Trnh t thu c ging vi: Rubella virus strain
RVi/LA.CA.USA/45.08/2B CRS, complete genome
Cc vng giu GC va virut u c th c trnh
t c (vic ny rt kh khi thc hin bng
my gii trnh t th h c)
chnh xc cao: 51782 coverage vi Q >30.