This document discusses genetic fingerprinting and DNA analysis techniques. It begins by describing different types of DNA markers that can be examined, including VNTRs, STRs, and RFLPs. It then outlines the basic steps of genetic fingerprinting, which includes sampling oral mucosa cells, lysing the cells, precipitating proteins and DNA, washing and drying the DNA pellet, and dissolving the DNA in water. The document provides details on how techniques like centrifugation and precipitation are used at different steps. It also mentions applications of genetic fingerprinting like paternity testing and PCR.
RNA, DNA Isolation and cDNA synthesis.pptxASJADRAZA10
Isolation, quantification of nucleic acids from wheat and synthesis of cDNA.
Introduction
List of Genotypes
DNA Isolation (CTAB method)
Qualitative check of DNA- Gel electrophoresis
Quantitative test of DNA- Spectrophotometer
Protocol for RNA Isolation
RNA Confirmation
Normalization of RNA
cDNA Synthesis
Protocol for DNA Isolation of plant
50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish.
Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer.
1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube.
The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins)
The tubes were cooled for 10 minutes in ice.
The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins.
After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added.
Then mixed gently for 5-7 mins by inverting the tubes.
Again centrifuged for 10 mins @10,000 rpm
After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight.
Following day, tubes were again centrifuged @10,000rpm for 10 mins.
The supernatant was discarded without disturbing the DNA pellet.
70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins.
Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded.
Pellet (DNA Precipitate) was air dried for 10 mins.
Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C.
1g of analytical grade Agarose was weighed.
100 mL of autoclaved 1X TBE was added in flask.
Now heated on the oven until the solution becomes transparent.
Solution was allowed to cool down to 60℃.
2 μL of Ethidium Bromide (EtBr) is added in the flask.
Melted agarose gel was poured into the casting tray along with comb.
Any bubble in the gel was removed.
After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank.
Once gel got solidified, it was transferred it into gel tank.
A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only.
Then the mixture (sample +loading dye) was loaded into the well.
Then electrophoretic unit was run at 90 volt for 50-55 mins.
After that gel was put into the Gel Doc to see the DNA band
(using UV light).
Bright colour band were observed as in the figure.
Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
Una delle attività svolte dai ragazzi del Liceo Scienze Applicate "M.Hack" presso il CREA di Turi nel laboratorio di Biologia Molecolare è stata quella dell'estrazione del DNA dalle giovani foglie di vite.
I ragazzi hanno seguito e realizzato con passione e coinvolgimento le attività, come dimostra il report da loro redatto in lingua italiana e inglese.
Un encomio ai ragazzi per l'eccellente lavoro svolto ed un ringraziamento particolare allo staff del CREA ed alla prof.ssa Cinzia Montrone per la supervisione dell'elaborato.
RNA, DNA Isolation and cDNA synthesis.pptxASJADRAZA10
Isolation, quantification of nucleic acids from wheat and synthesis of cDNA.
Introduction
List of Genotypes
DNA Isolation (CTAB method)
Qualitative check of DNA- Gel electrophoresis
Quantitative test of DNA- Spectrophotometer
Protocol for RNA Isolation
RNA Confirmation
Normalization of RNA
cDNA Synthesis
Protocol for DNA Isolation of plant
50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish.
Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer.
1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube.
The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins)
The tubes were cooled for 10 minutes in ice.
The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins.
After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added.
Then mixed gently for 5-7 mins by inverting the tubes.
Again centrifuged for 10 mins @10,000 rpm
After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight.
Following day, tubes were again centrifuged @10,000rpm for 10 mins.
The supernatant was discarded without disturbing the DNA pellet.
70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins.
Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded.
Pellet (DNA Precipitate) was air dried for 10 mins.
Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C.
1g of analytical grade Agarose was weighed.
100 mL of autoclaved 1X TBE was added in flask.
Now heated on the oven until the solution becomes transparent.
Solution was allowed to cool down to 60℃.
2 μL of Ethidium Bromide (EtBr) is added in the flask.
Melted agarose gel was poured into the casting tray along with comb.
Any bubble in the gel was removed.
After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank.
Once gel got solidified, it was transferred it into gel tank.
A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only.
Then the mixture (sample +loading dye) was loaded into the well.
Then electrophoretic unit was run at 90 volt for 50-55 mins.
After that gel was put into the Gel Doc to see the DNA band
(using UV light).
Bright colour band were observed as in the figure.
Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
Una delle attività svolte dai ragazzi del Liceo Scienze Applicate "M.Hack" presso il CREA di Turi nel laboratorio di Biologia Molecolare è stata quella dell'estrazione del DNA dalle giovani foglie di vite.
I ragazzi hanno seguito e realizzato con passione e coinvolgimento le attività, come dimostra il report da loro redatto in lingua italiana e inglese.
Un encomio ai ragazzi per l'eccellente lavoro svolto ed un ringraziamento particolare allo staff del CREA ed alla prof.ssa Cinzia Montrone per la supervisione dell'elaborato.
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis .docxmoirarandell
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis
2
Objective
· Digest DNA of pGLO plasmid using restriction endonuclease enzymes.
· Run an agarose gel to separate the DNA fragments.
Background
Restriction enzymes cut DNA at specific sites generating a number of different sized fragments. The size of the fragments will depend on the number of sites the plasmid has and the specific enzyme used. The number of fragments can be predicted by viewing the map of the plasmid
Gel electrophoresis is a means of separating DNA in an electrical field. DNA is negatively charged and so will move to the anode (+). Larger fragments will move slower through the agarose matrix than the smaller molecules. Agarose is a polysaccharide polymer derived from seaweed: it is a purified from agar by removing the agaropectin component. Fragments are visualized using ethidium bromide, which will glow orange when exposed to UV light.
Materials
Restriction digest
· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs) – (KEEP ON ICE)
· Plasmid prepared in lab 7
· NanoDrop Lite spectrophotometer
· Microfuge tubes – Sterile
· 37 C degree bath – block heater
· Sterile 10ul and 200ul tips
· Bleach bottles for cleaning bench
· 10X NE Cut Smart Buffer – comes with enzyme
· Nitrile gloves
· Sterile DI water
· Shaved ice
· Ice block for enzymes
Gel Electrophoresis
· Agarose
· Sterile miliQ Water
· 15 well comb
· 50x TAE buffer
· DNA ladder – diluted in sample buffer (1 KB)
· Gel loading dye
· Gel electrophoresis chamber
· Power supply
· Ethidium bromide
· Gel Sys – visualization system
_______________________________________________
Procedure
Restriction Digest of plasmid DNA
· Safety: Wear nitrile gloves – prevent DNAase from your hands affecting the reaction and protect yourself from ethidium bromide
· Clean the bench with bleach - prevents exogenous enzymes interfering you’re your digests.
· Use the NanoDrop to determine the amount of DNA in your plasmid prep. Use this information to calculate how much sample you need to pipette into the reaction mix.
· Label an Eppendorf tube ‘+’ and another ‘-‘
· Make up a reaction mix in both tubes as follows for one of your plasmid samples
· add 1ug of DNA from your plasmid prep
· 5ul of 10X NE Cut Smart Buffer
· Sterile DI water to make the reaction mix to 50ul
For the + tube
· DNA
x ul
· 10X NE Cut Smart Buffer
5ul
· Nhe1 (add last to + tube)
1ul
· EcoR1 (add last to + tube)
1ul
· Sterile DI water
To make final volume to 50ul
· Add the restriction enzymes last to the + tube ONLY
· Repeat with the other two plasmid samples
For the – tube
· DNA
x ul
· 10X NE Buffer
5ul
· Nhe1
None
· EcoR1
None
· Sterile DI water
To make final volume to 50ul
· Do not add any enzyme to the ‘-‘ tube
· Repeat with the other two plasmid samples
· Mix the tubes by flicking – DO NOT VORTEX
· Give a 5 second spin in the centrifuge to bring the contents to the bottom
· Incu.
DNA Extraction, DNA quantity-quality check & Amplicon quantity checkMohiuddin Masum
DNA Extraction using Reliaprep™ blood gDNA extraction protocol
DNA quantity-quality check using Nanodrop and
Amplicon quantity check using Fluorometer
DNA extraction procedure video YouTube link
https://www.youtube.com/watch?v=uk2H4tJUAto
Nanodrop procedure video YouTube link
https://www.youtube.com/watch?v=JlMuF0FAU1g
Fluorometer procedure video YouTube link
https://www.youtube.com/watch?v=To1vSP1bNxo
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis .docxmoirarandell
Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis
2
Objective
· Digest DNA of pGLO plasmid using restriction endonuclease enzymes.
· Run an agarose gel to separate the DNA fragments.
Background
Restriction enzymes cut DNA at specific sites generating a number of different sized fragments. The size of the fragments will depend on the number of sites the plasmid has and the specific enzyme used. The number of fragments can be predicted by viewing the map of the plasmid
Gel electrophoresis is a means of separating DNA in an electrical field. DNA is negatively charged and so will move to the anode (+). Larger fragments will move slower through the agarose matrix than the smaller molecules. Agarose is a polysaccharide polymer derived from seaweed: it is a purified from agar by removing the agaropectin component. Fragments are visualized using ethidium bromide, which will glow orange when exposed to UV light.
Materials
Restriction digest
· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs) – (KEEP ON ICE)
· Plasmid prepared in lab 7
· NanoDrop Lite spectrophotometer
· Microfuge tubes – Sterile
· 37 C degree bath – block heater
· Sterile 10ul and 200ul tips
· Bleach bottles for cleaning bench
· 10X NE Cut Smart Buffer – comes with enzyme
· Nitrile gloves
· Sterile DI water
· Shaved ice
· Ice block for enzymes
Gel Electrophoresis
· Agarose
· Sterile miliQ Water
· 15 well comb
· 50x TAE buffer
· DNA ladder – diluted in sample buffer (1 KB)
· Gel loading dye
· Gel electrophoresis chamber
· Power supply
· Ethidium bromide
· Gel Sys – visualization system
_______________________________________________
Procedure
Restriction Digest of plasmid DNA
· Safety: Wear nitrile gloves – prevent DNAase from your hands affecting the reaction and protect yourself from ethidium bromide
· Clean the bench with bleach - prevents exogenous enzymes interfering you’re your digests.
· Use the NanoDrop to determine the amount of DNA in your plasmid prep. Use this information to calculate how much sample you need to pipette into the reaction mix.
· Label an Eppendorf tube ‘+’ and another ‘-‘
· Make up a reaction mix in both tubes as follows for one of your plasmid samples
· add 1ug of DNA from your plasmid prep
· 5ul of 10X NE Cut Smart Buffer
· Sterile DI water to make the reaction mix to 50ul
For the + tube
· DNA
x ul
· 10X NE Cut Smart Buffer
5ul
· Nhe1 (add last to + tube)
1ul
· EcoR1 (add last to + tube)
1ul
· Sterile DI water
To make final volume to 50ul
· Add the restriction enzymes last to the + tube ONLY
· Repeat with the other two plasmid samples
For the – tube
· DNA
x ul
· 10X NE Buffer
5ul
· Nhe1
None
· EcoR1
None
· Sterile DI water
To make final volume to 50ul
· Do not add any enzyme to the ‘-‘ tube
· Repeat with the other two plasmid samples
· Mix the tubes by flicking – DO NOT VORTEX
· Give a 5 second spin in the centrifuge to bring the contents to the bottom
· Incu.
DNA Extraction, DNA quantity-quality check & Amplicon quantity checkMohiuddin Masum
DNA Extraction using Reliaprep™ blood gDNA extraction protocol
DNA quantity-quality check using Nanodrop and
Amplicon quantity check using Fluorometer
DNA extraction procedure video YouTube link
https://www.youtube.com/watch?v=uk2H4tJUAto
Nanodrop procedure video YouTube link
https://www.youtube.com/watch?v=JlMuF0FAU1g
Fluorometer procedure video YouTube link
https://www.youtube.com/watch?v=To1vSP1bNxo
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
5. Sequences (Markers) examined in DNA
fingerprinting
• VNTRs-variable number tandem repeats (mikrosatelite); composed of 8-80
bp repeat units (e.g., [GCGCAATG]n) which are tandemly repeated so that the
overall length is 1-30 kb
• STRs-short tandem repeats; composed of 2-7 bp repeat units (e.g., [AC]n)
which are tandemly repeated so that the overall length is less than 1 kb
• RFLPs- restriction fragment length polymorphisms.
11. 1. Sample of Oral Mucosa (kumur-kumur dg 8 ml akuades).
The water contains cells from oral mucosa, enzyme.and whatever
you have in your mouth. We want to get hold of the cells
2. Add 2 ml of this solution into eppi (tube) with pipette and put it into
centrifuge for 2 minutes at 3200rpm.
During centrifugation, the cell move outwards due to their weight.
After this process u can see a small white spot at the bottom of
your eppi, called pellet. These are your cell.
3. Pour away supernatant. To guarantee that
u have enough cell material,
repeat the step 2 at least two times.
What the functionally of centrifuge?
Step 1: Sampling from oral mucosa
12. Step 2: Lysis of the cells
• Add 500 uL Lysis buffer L to the pellet with pipette. Snip
your finger against eppi until the pellet has disappeared.
One ingredients of the lysis buffer is a detergent,
which dissolves the cells.
How does the lysis buffer work?
13. Step 3: Precipitation of
Protein
• Add 100uL precipitation buffer F, shake the eppi well and
put in on ice for 5 minutes.
The precipitation buffer contains salt (potassium acetate)
with which proteins are precipitated.
• After that, centrifuge the eppi for 15 min at 20000rpm to
palletize the precipitated proteins
14. Step 4: Precipitation of DNA
• Ambil 400uL supernatan ke eppi baru. Add 360uL
isopropanol. Taruh kembali eppi ke dalam es selama
beberapa menit.
• Shake well and centrifuge again (20000rpm, 15 min)
Centrifugation causes pelleting
of DNA at the bottom of the eppi.
15. Step 5: Washing the DNA
• Carefully pour away the supernatant and add 500uL
70% cold ethanol and put back in the centrifuge
(20000rpm, 5 min)
After pouring away again, u can see a small white pellet
at the bottom of the tube.
The pellet is then dried at 60oC in heating block (leave
the eppi open)
16. Step 6: Solution of DNA in Water
• Dissolve the pellet in 30uL UV-water.
UV-water has been irradiated with ultraviolet light. Any DNA that
may have been in the water has been destroyed.
New eppi only contains
your DNA,
solved in UV-water ☺
23. References
Godbey WT. 2014. An Introduction to Biotechnology: The science, technology and
medical applications. Elsivier, Tulane University, New Orleans, Louisiana.
Lawrence Kobilinsky, Louis Levine, Henrietta Margolis-Nunno. 2007. Forensic DNA
Analysis. Infobase Publishing
Science Bridge Module. 2010. Bridging University to High School: In real Modern
Biology. Bogor Agricultural University (IPB) in Collaboration with University of
Kassel Germany.
Editor's Notes
# # Asslamualaikum wr wb. Alhamdulillah wasolatu wasslamu ala rosulillah ala alihi wa sohbihi ajmain.
Robbi srohlisodri wayassirli amri wahlul uqdatammillisani yafqahu qauli.
# Marilah kita lanjutkan kembali kuliah bioteknologi. Minggu ini kita akan membahas topik Gentic Fingerprint.
Istilah genetic fingerprint mungkin sangat familiar kita dengar, Apa yang dimaksud dengan genetic fingerprint bagaimana sebenarnya teknik Gentic Fingerprint ini? Itu yang akan kita bahas dalam kuliah kali ini.
Materi kuliah yang saya gunakan adalah modul praktikum yang digunakan untuk siswa SMA. Melalui program Science Bridge Bridging University to High School: In real Modern Biology. Kolaborasi antara IPB, Untirta dan beberapa universitas di Indonesia dengan University of Kassel Germany. Dan beberapa buku text yang bs kalian temukan pada akhir slide.
# Genetic atau DNA Fingerprint adalah bioteknologi yang digunakan untuk mengetahui identitas seseorang.
Teknologi ini banyak digunakan oleh bagian forensic kepolisian sebagai bukti pendukung untuk membantu penyidik dan pengadilan, apakah tersangka bersalah atau tidak.
#DNA fingerprint juga digunakan untuk test paternity, paternitas secara bahasa artinya garis ayah.
singkatnya paternity test ini digunakan untuk mengetahui siapa orang tua orang yang ingin tes.
Misalnya jika ada bayi yang tertukar, kasus penentuan harta warisan terhadap turunan yang sah, seseorang mengklaim ayah atau orang tua dari si anak dll. Ini seperti sinetron atau drama heee Banyak scenario lah ya.
#
Dimana kita bisa menemukan DNA pada manusia? Ini jawaban yang harusnya bisa dijawab dengan mudah oleh mahasiswa semester 6, yaitu disemua sel tubuhnya. Juga pada mamalia lain, sapi, babi, semua sel mamalia mengandung informasi genetik.
# Dalm tubuh kita ada sekitar 10 pangkat 14 sel. Misalnya pada sel-sel saraf, sel-sel otot, atau sel-sel oral mukosa dalam mulut kita.
# Sekarang bayangkan kalian adalah detektif atau polisi yang menemukan bercak darah berceceran ditempat kejadian perkara sebuah tidak kejahatan, wahhh yang sering nonton Crime Investigation atau NCIS udah tahu banget pasti bs dibayangnkan seperti apa crime scene nya ini.
Kalian sebagai detektif perlu mengambil sampel darah sebagai sumber DNA untuk forensic di Lab. Sumber DNA dari sel-sel lain juga dapat diambil seperti cairan sperma, tulang, kulit, cairan saliva ludah, urine, feses maupun sel-sel pada folikel rambut. Sampel ini kemudian di bawa ke laboratorium lalu diisolasi DNAnya untuk dilakukan analalisis lanjut. Nti kita akan kita bahas.
# Meskipun berasal dari sel-sel berbeda. Semua informasi didalam sel-sel tersebut adalah sama dalam satu individu. Namun apakah semua informasi dalam sel itu berguna ? Jawabannya adalah TIDAK.
# Tentunya kita sudah belajar apa yang dimaksud dengan gen?
# Pada E coli, genome E coli sekitar 4,6 juta pasang basa, memiliki gen sekitar 4500
# Pada manusia genome kita sekita 3 milyar pasang basa, dengan estimasi 25 000 gen
# Pada organisme triploid, disini gandum contohnta triticum aestivum genomenya sebesar 17 milyar pasang basa, dengan jumlah gen diperkirakan 50 000.
# Nah ini ada kalkulasi menarik, sebuah perbandingan yang dapat menggambarkan seberapa banyak informasi yang kita miliki di genome dalam sel-sel kita.
# Jika kita memiliki 3 milyar pasang basa, bayangkanbasa-basa dalam DNA kita ini adalah huruf2 yang akan kita tulis dalam buku. Ada berapa bayak buku yang bs ditulis?
Jika Satu halaman berisi, 4800 huruf, berarti 4800 pasang basa. Jumlah pasang basa yang ada dalam genome kita 3 milyar, berarti 3 milyar dibagi 4800 sama dengan 625 000. artinya ada 6500 halaman.
Jika buku yang ingin kita buat masing2 terdiri dari 300 halaman, berarti 62500 dibagi 300 sama dengan 2084 buku.
Dalam satu Rak buku dapat diisi 25 buku, sehingga 2084/25= 84 rak buku. Sebanyak itu lah informasi dalam DNA kita jika kita tulis dalam buku, ada 84 rak buku yang masing2 berisi 25 buku informasi genetik kita. Subhanallah ya…. Amazing huh..
# Ok, sekarang kita sudah py gambaran betapa banyaknya informasi yang dalam DNA kita. Pertanyaan selanjutnya, Bagaimana kita mempelajari informasi tsb. Bagaimana kita bs membaca dan memahami buku2 tsb?
Kita telah tahu bahwa ada sekitar 25 500 gen, GEN ini adalah region atau bagain dalam DNA yang dapat kita pelajarai dengan baik, karena gen ini adalah penyandi asam amino penyandi sifat.
Region yang menyandikan gen dalam DNA hanya sekitar 1,5% dari total genome kita. Bagaimana dengan sisanya? 98,5% lainnya, yang jumlahnya sebagian besar komposisi DNA kita.
Region ini belum banyak dipahami fungsinya, biasanya disebut “Junk DNA” sebetulnya istilah junk DNA ini menurut sy kurang cocok, kesannya gak berguna. Padahal kita saja yang belum tahu fungsinya, manusia belum mampu menguak misteri pada bagian ini .
Untuk mempelajari DNA kita butuh penanda atau marker. Gen2 td itu adalh marker, selain gen ada region yang memiliki ciri2 tertentu itu juga digunakan sebagai marker.
Bayangkan sebuah peta buta, untuk memahaminya kta butuh penanda kan, misalnya jembatan, jalan raya, hotel, atau gedung2. begitu jg dg DNA. Markers yang bukan Gen ini yang digunakan untuk analisis DNA fingerprint.
Diantara marker2 yang dapat dipakai u DNA fingerprint adalah, VNTR-variable tandem number tandem repeat.
VNTR merupakan region DNA yang terdiri dari 8-80 repeat atau pengulangan misalnya GCGCAATG berulang 20 kali, pengulangan ini biasanya terjadi dari 1-30kb.
Marker lain nya, STR- shorts tandem repeat, bisanya pengulangannya lebih pendek 2-7 bs sepanjang 1 kb.
Yang terakhir RFLP-restriction fragment lenghth polymorphism. RFLP dibuat dengan cara memotong2 genome DNA dg enzim restriksi sehingga dihasilkan fragmen dna dengan panjang yang berbeda-beda, kemudian di running pada gel elektrphosesis.
Contohnya ada pada gambar, Gambar A adalah RFLP pattern hasil analisis sampel darah dari tempat kejadian. Ada 2 suspect atau tersangka, sampel dr tempat kejadian dan Officer Smedley.
Gambar B. jika kita hilangkan pita-pita yang common atau sama yang ada pada sampel, korban dan tersangka. Kita akan melihat bahwa pada suspect 1 ada satu pita yang tidak ditemukan pada crime scene, sedangkan pada suspect 2 ada pita yang hilang atau tidak muncul pada crime scene.
Sedangkan officel Smedley memenuhi semua kriteria. Apakah Officer Smedley pembunuhnya, yang kemungkinan sangat diragukan, atau cara pengambilan sample yang ia lakukan tidak benar?
Dalam kasus ini Kedua tersangka dinyatakan tidak bersalah dan dibebaskan.
# Paternity test memungkinkan kita menganalisis possibility beberapa orang yang mengaku sebagai ayah dari seorang anak.
# VNTR sampel orang-orang tersebut kemudian dibadaningkan dengan VNTR si anak.
# The biological father atau ayah yang sebenarnya pasti memiliki joint allel atau alel-alel yang sama dengan si anak.
# Pada paternity test marker yang digunakan umumnya VNTR atau microsatelite.
D1S80-locus pada chromosome nomor satu digunakan sebagai marker.
Marker ini terdiri dari 16 bp-DNA sekuense yang berulang beberapa kali.
Jumlah pengulangan bisa bervariasi dalam tiap individu
Sehingga alel pada lokus tersbut berbeda-beda panjangnya.
# Misalnya pada gambar terdapat 2 alel berbeda, berbeda alel 1 lebih pendek dibanding alternative alel 2 yang lebih panjang 1 kali pengulangan.
# Perhatikan gambar berikut mikrosatelit lokus D1S80 milik Anna dan Otto
# Anna memiliki satu jenis alel karena alel yang berasal dari ayah dan ibunya sama.
Sedangkan oto punya 2 macam alel
# ada berapa genotype yang ada pada lokus D1S80?
Sampai saat ini ada 28 alel berbeda.
16 bp sekuesne diulang antara14 sampai 41 kali. Jumlah genotype yang mungkin ada 406, lihat rumus bagaimna mengkalkulasinya.
# Sekarang bagimana kita mengamplifikasi lokus D1S80?
# Step pertama yang dilakukan adalah mengisolasi DNA. Sampel DNA dapat diambil dari sel-sel oral mukosa. Untuk praktikum sederhana, cara yang dilakukan adalah dengan berkumur2 selama 5 menit kemudian air kumur tsb digunakan sebagai sumber sampel.
# ambil 2mL cairan air kumur lalu sentrifuge dengan kecepatan tinggi. Pellet yang dihasilkan didasar tube adalah sel-sel mukosa.
# Langkah selanjutnya, larutkan pellet sel dengan lusu buffer, snip dengan jari atau menggunakan pipet agar pellet tersuspensi dengan baik dengan buffer. Tandanya adalah endapan pellet sudah menghilang dari tube.
# Tambahkan buffer presipitasi, shake sebntar lalu taruh dalam es 5 menit. Setelah itu sentruifuge dengan kecepatan super tinggi 20000 rpm selama 15 menit. Pellet yang mengendap didasar tube adalah protein yang menggumpal.
Dimana DNA? DNA ada lapisan supernatant, pada fase ini penting sekali untuk memipet supernatant dengan baik sehingga pellet tidak ikut terpipet.
# Step selanjutnya adalah presipitasi DNA. Pipet 400uL supernatant pindahkan ke tube bary lalu tambahkan isopropanol dingin. Shake well dan sentrifuge dengan kecepatan sama selama 15 menit.
Pellet DNA akan terlihat seperti titik dibagian bawah tabung
# Pencuncian DNA dapat menggunakan etanol dingin 70% dicuci dua kali, lalu pellet DNA dikeraingkan pada heating lock.
# DNA ini kemudian dapat di larutkan dalam TE buffer atau DNAse free water atau UV water. Sebagai stock DNA.
# DNA stick kita adalah Template DNA yang akan digunakan untuk amplifikasi Lokus D1S80.
# Perbanyakan DNA menggunakan PCR, mix PCR solution dengan komposisi template DNA yang diisolasi dari oral mukosa, sepasang primer Lokus D1S80, nukleotida dan taq polymerase .
# Setelah selesai, amplicon atau PCR produk di running di agar gel electrophoresis.
# Beginilah hasilnya. Kolom paling kanan M, adalah marker DNA 3000 1000 500 dan 200 bp. Kolom 1 sampai 16 adalah 16 sampel berebda dari orang yang di tes pada praktikum.
# Contoh paternity test misalnya, ada dua orang yang mengaku menjadi ayar dari anak Mary, yaitu Bob dan Larry dengan profil VNTR spt pada gambar. Siapakah ayah biologis anak Mary?
# Dari hasil tes pada gambar, dan penjelasan sebelumnya, siapakah ayah dari anak Mary?
Amati pita2 VNTR yang ada pada gambar, pattern VNTR anak seharusnya memiliki kombinasi ayah dan ibunya.
# Demikian kuliah kali ini. Silahkan manfaatkan waktu yang ada pada kuliah online untuk bertanya dan berdiskusi.
Semoga bermanfaat wasslamualaikum wr.wb.