genetic engineeringparasitology.sbmu.ac.ir/uploads/...presentation1.pdf · molecular weight (mw)...
TRANSCRIPT
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سید طبائی
دارد و آن آگاهی است يك فضیلت وجىد رد جهان تنها است جهل آن و ؛ گناه يك تنها و
مىالان-عارف زبرگ
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Genetic engineering Calculations for Molecular Biology and
Biotechnology
Frank H. Stephenson
Molecular cloning A laboratoay manual
Sambrook and Russell
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Prefix choice should result in >1 and is < 1000 0.00000005kg = 50 ng no 0.05 ug 0.0025 ml =2.5x 10-3 ml = 2.5ul 0.03 ug = 3x 10-2 ug = 30 ng
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Solution W/W: g/g (Lactophenol)
Solution W/V: g /ml(sugar solution)
Solution V/V:ml/ml (alcohol )
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Molecular weight (MW) =sum of its atomic weights
Pure /MW with water:
Normal concentration : amount of soluble material g/L
Mol:equivalent to 6.023 x 1 023 molecules
Molarity M: MW(g)/L (exactly 1L)
Molal concentration (m): >1L
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The concentration coefficient X
Storage >1X
Working = 1X
Percent solution
Percent of the amount(g/100cc), (cc/100cc)
EX. 200cc Etanol 95%(0,95x200=190 )
190cc absulat Alcohol + 10cc water
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Unit volume
1 Liter = 10 dcL =100 cL
100 dc3
Cm3 =cc =ml=(10x10x10) mm3
mm3 =ul
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Converting molarity to percent =
Converting percent to molarity=
Normality= M/Capacity MW
Amount at 100 x10
10
M X MW
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Working solution prepared
Direct proportion
Increase both or Reduced both (M1V1≠M2V2)
Inverse proportion
Increase one with Reduced Other(M1V1=M2V2)
TE buffer 50 cc (Tris 10 Mm و EDTA 1 Mm )
Stock (Tris 1M ,EDTA o.5M)
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Centrifugation
nomogram
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Biological materials used for DNA profiling
Blood
Hair
Saliva
Semen
Body tissue cells
vaginal cells
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DNA extraction
Step1:Sample preparation:
BLOOD : EDTA (1mg/ml)
Sputum :20 ‘/95oc
Vaginal Swab;
Swap: 2 hours in dry air
In 200 ml of distilled water
30’/56oc and 10’/95oc
Tissue
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Methods of physical and mechanical
Grinding: with a mortar and pestle Chinese
Freezing and thawing( membrane failure)
shock thermal
Sonication
chemical (enzymes and some detergents)
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Genetic engineering
step2
◦ Disrupt cell
Animal cellular SDS
Plant cellular pectinase /cellulase
Bacteria + lysozyme (peptidoglycan)
Bacteria - lysozyme& EDTA
(lipopolysaccharide)
◦ Buffer(Tris)
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Step3
Separation of DNA
RNA
Liped
Glucose
Protein
……
Ion-exhange chromatogaraphy
Enxymatic digestion & organic solvents
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DNA Extraction :
Cell lysis buffer(STE)
Tris (10mM,pH 8): buffer
EDTA (1mM): Inhibition of DNase
NaCl 100mM : physiologic
SDS 1%: Dissolve Lipid
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Proteinase K:
Endonuclease: peptide bonds in the carboxylate
Aliphatic(linear),(aromatic)ring,hydrophobic
enzyme's stability 4-12.5 (optimal PH is 7.5 – 8).
temperature (20 to 65oc ) optimal 37oc
Concentrations: 50-100 μg/ml
Remove DNases and Rnases
activation of the enzyme: SDS 1%
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The removed organic materials:
Phenol + chloroform 1/1
Denaturation of proteins (phenol / pH >7)
DNA&RNA(White layer between the
aqueous phase & phenol) Ribonuclase
remove phenol (Chloroform )
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Removed salts
concentrate the DNA:Sodium acetate
The DNA is ionized (Reduced solubility in non-
aqueous media
High purity alcohol :The insoluble DNA
Washing: ethanol 70
Drying, DNA dissolved in distilled water
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Nucleic acid quantification
ultraviolet(uv)spectroscopy
Pure DNA A260/A280 ratio close to 1.8.
phenol or protein contamination < 1.8.
RNA is present in the DNA prep >1.8
Pure RNA ratio close to 2.0
Absorbance and optical density (OD)
1 OD of dsDNA = 50ug/mL
1 OD of ssDNA = 33ug/mL
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Calculating DNA concentration as a mM mount
Calculate Molarity solution DNA
The Absorbtion coefficient (E260) for a solution 1
mM solution of dsDNA is 6.7
solution of ssDNA is 8.5
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resuspended the DNA in a volume of 50 ul.
dilute 20 ul into a total volume of 1000 ul distilled water.
Absorbance 260= 0.550 ; 280=0.324.
DNA concentration of the 50 uL plasmid prep(1.35ug/ul)
What is the A260/280 ratio(0,55/0.324) of the purified
DNA? 1.7
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:روش کار
نمونه +µl 500 (ورتکس ونيمساعت در دمای محيط)کننده بافر ليز
µl2 پروتئينازK (20mg/ml)(h1 C /°55 15’و/C °80 )
(.12000دقيقه سانتريفوژ 2ورتکس و)محلول فنل بازي هم حجم
مايع روييDNA(4 اليه, DNAرسوب ، اليه نازک پروتئين، فنل و)
اضافه کردن هم حجم کلروفرم( سانتريفوژ دقيقه 2ورتکس وrpm 2000)
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مايع رويي DNA (3 اليه در صورت شفاف نبود مايع مرحله قبل تکرار
(شود
1/10 محلول رويي استات سديمM3 برابر حجم الکل مطلق 2و
10‘سانتريفوژ ,ورتکسC °5 /rpm 12000
دو بار(حذف استات سديم ) 70شستشو با الکل
37/ ’10خشک کردن رسوبoc
آب مقطر استريل اضافه ودر فريز نگهداری شود
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sizing DNA fragments by
gelelectrophoresis
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POLYMERASE
CHAIN
REACTION
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PCR (technique that takes a specific sequence of DNA) fotocopy
Kary Mullis
1983 (PCR was invented )
1985 (The first published)
1993 (the Nobel prize for Chemistry )
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DNA
Example of bonding pattern.
Primary strand
CCGAATGGGATGC
GGCTTACCCTACG
Complementary strand
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WHAT IS IN THE PCR REACTION ?
Template DNA
Primers
Reaction buffer )Tris, Ammonium and/ or Potassium ions, Magnesium ions, Bovine serum albumin ]
Nucleotides [ dNTPs ]
DNA Polymerase
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Primer design (temperature calculation)
Melting temperature (Tm) is temperature at
which the primer will dissociate.
Annealing ) should be just 1-2 C below Tm.(
Both primers should have approximately the same
Tm value.
Tm estimation:
Tm = (4 x [G+C]) + (2 x [A+T])ºC
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Preparation Primer
Shelf life of oligonucleotide
• Acidic PH values (Sensitive) MWG-Biotech (PH-8-8/5)
• Nucleases(Danger of degradation)
• suitable hygiene measures at the workplace
– aid by salts two & three-valent ions Low amounts of EDTA
• MWG-Biotech 10-20mM tris- EDTA
• cinnaGen :purest water
• SSDNA(less stable in freezing &thawing)
– Kept to a minimum
– Shorter intervals(1-2days) aliquot at 40c
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• SSDNA(less stable in freezing &thawing)
– Kept to a minimum
– Shorter intervals(1-2days) aliquot at 40c
Short-term
◦ Aliquots should be prepared and stored at -200c
is not an adequate for the exclusion of enzymatic reaction
Long-term( >1/2years)
◦ Recommened to store at -800c
◦ Up to two years
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OD(optical Density)
MW
Length
Tm(melting temperature)
GC content
Volume for 100 pmoles/ul(uM):
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Primer :
Solution( ug)
Lyophilized ( pmol)=
OD Mw Pmol uM/ul(500ul) 50ul work stock(10uM/ul)
6 8201 21948 43.9 11.39
Pmol.ul=uM
Primer work:10 um/ul Necessary amount :./1-1 ul
Standard reaction :./4-./5 ul
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Short-term
◦ Aliquots should be prepared and stored at -200c
is not an adequate for the exclusion of enzymatic
reaction
Long-term( >1/2years)
◦ Recommened to store at -800c
◦ Up to two years
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What is Taq polymerase?
the DNA polymerase I for Thermus aquaticus
half life : 95oc >40
Many of its enzymes (including DNAP I) will not
denature at high temperatures
PCR
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انواع پلی مرازها
حساس به حرارت
Klenow fragment
T4 DNA polymerase
مقاوم به حرارت
Tag polymerase
Ampli tag
Hot start tag
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• DNA polymerase • Strings of DNA synthesis on template strings
»dNTP »Primer
• DNA polymerase I :
» Exonucleases activity 3’→ 5` proof reading(Klenow)
» DNA polymerase activity 5`→ 3` »activity RNase H
• Application: • DNA Labeling Nick translation • Created ends Blunt
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dNTP MIX
10mM of each (dATP,dCTP,dGTP,dTTP)
PH:7.5
Store:-20oc
Shipment :Wet ice
Final concentration: o.2mM each/ul
.2x100=20 (2ulx/100ul reaction)
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Tag DNA polymerase
Concentration :5 unit/ul
Store:-20oc
Shipment :Wet ice
Final concentration: o.15 ul
2.5ul/100ul reaction
Mgcl2 (50mM)
Final concentration: 1.5mM
1.5x100=150 (3ul/100ul reaction)
PCR buffer:10x Final concentration:1x
1x/100ul reaction=100÷10ul =10ul
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Typical PCR mix
In a thin wall Eppendorf tube assemble the following
PCR components Amount
Template DNA (5-200 ng) : 1 µg
1 mM dNTPs (200 uM final)
10 X PCR buffer(pH 8.3-8.8)
25 mM MgCl2 (1.5 mM final)
20 uM primer(20 pmoles final)
5 units/uL Taq DNA polymerase(1.5 units)
Water
Final Volume
variable
10 uL
5 uL
3 uL
1 uL
0.3 uL
variable
50 uL
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:
Ramp timeزمان بين دو مرحله برای تغييردما درترموسايکلررا گويند
محصول سکانس هدف که درانتهای دارای سکانس پرايمراست: Amply cone
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THE BASICS OF PCR CYCLING
30 TO 35 CYCLES EACH COMPRISING OF :
Denaturation ( 94° C ) for 30 seconds
Annealing ( 54-60° C ) for 30 seconds
Extension ( 72° C ) – Time depends on product size
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فرايند تقليب) denaturing (
باز شدن دو رشتهDNA با کمک حرارت با شکستن پيوند هيدروژنی
(ثانيه 30اکثرا )درجه 92-100معموال حرارت
فرايند اتصال(Annealing)
اتصال پرايمر به الگو با کاهش درجه حرارت بر اساس قانون واتسون و گريک
درجه کمتر از 3-5دمای اپتيموم معموالTM ثانيه است 30است زمان حدود
فرايند طويل شدن(extention)
گسترش پرايمر بر روی رشته الگو با کمک آنزيم پليمراز در حرارت مناسب
دما آنزيمDNA polymerase حدودoc72 است (1000هرbp حدود يک دقيقه)
PCRمراحل
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PCRسيکل در
محصول طول مشخص ندارد و سنتز تا زمان توقف و سيکل بعد ادامه دارد:و دوم اول
(طول مشخص )قطعاتی از توالی بازها از الگوی هدف سنتنز می شود : سيکل سوم
تکثير تصاعدی توالی باز های :سيکل چهارمDNA
محاسبه تعداد کپی ها
(2n -2n)X N= تعداد سيکل هاX=تعداد نسخه از الگوی اوليه
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عوامل محدود کننده
سيکل 25-35غير فعال شدن آنزيم بعد از
جزء کلنوDNA polymerase 1 به حرارت حساس و بايد درهرسيکل اضافه شود
افزايش غلظت رشته هدف و رقابت با پرايمر(Reannealing)
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PCR Cycles
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PCR Cycles
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PCR Cycles
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PCR Cycles
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PCR Cycles
3” 5”
3”
3”
3”
5
”
5”
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Enhancers of PCR
Formamide 5%
DMSO <10%
PEG 5-15%
Glycerol 10-15%
Tween20 0.1-2.5%
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Conventional PCR
Nested-PCR
RT PCR :(Reverse Transcriptase-PCR)
Real time PCR:
Multiplex-PCR
ARMS-PCR(Amplification Refractory Mutation System)
Touchdown PCR
Asymmetric PCR
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