The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA. First described in 1985, it was made possible by the discovery of Taq polymerase. The primary reagents used in PCR are DNA nucleotides, template DNA, primers, and DNA polymerase.
What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References
What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References
Polymerase chain reaction (PCR) is a technique in molecular biology used to
amplify (multiply) a single copy or a few copies of a piece of DNA, generating
thousands to millions of copies of that particular DNA sequence.
PCR is a revolutionary molecular biology technique used for enzymatically replicating DNA . This technique allows a small amount of DNA molecule to be amplified many times in an exponential manner . It is commonly used in medical and biological research labs for variety of tasks such as detection of hereditary disease , identification of genetic fingerprints diagnosis of infectious disease , cloning of genes and paternity testing .
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies . The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR.
There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95°C. For example, the half-life of Taq polymerase at 95°C is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100°C. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.
At 75°C, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute!
Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Direct PCR cloning methods include TA and GC cloning, as well as TOPO® Cloning, and enable direct cloning of PCR fragments. For example, the TA cloning approach takes advantage of the 3’ A overhang naturally added to products by Taq polymerase following PCR. The resulting sticky ends then enable recombination with DNA fragments containing 3’ T overhangs, such as linearized vectors.
During indirect PCR cloning, the PCR products are modified prior to recombination with other DNA sequences. For example, in restriction cloning, restriction sites are frequently introduced via PCR to enable restriction digestion and ligation with linearized vectors. PCR mutagenesis is a technique used to generate site-directed sequence changes such as base substitutions, inserts and deletions.
To insert a single point mutation via mutagenesis, for example, PCR primers are designed that contain the desired base change, usually in the middle of the primer sequence. PCR is then performed with the mutagenic primers and a high-fidelity DNA polymerase, which results in the incorporation of the desired mutation into the original sequence.Allele-specific PCR is used to detect sequence variations and ultimately determine the genotype of an organism.
For allele-specific PCR, primers are designed to flank the region of interest. The most common application of PCR is gene expression analysis
this ppt contain about pcr technique and its three process,primers in pcr,dna polymerase in pcr,melting temp of dna in pcr and applications of pcr technology
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.
PCR (polymerase chain reaction) and Extraction of DNA from fungal plant patho...AjayDesouza V
PCR, Polymerase chain reaction, types of PCR, Template DNA, DNA polymerase, Primers, Nucleotides (DNTPs or deoxynucleotide triphosphates ), Denaturation, Annealing, Extension, Types of PCR, Multiplex PCR.
Long-range PCR.
Single-cell PCR.
Fast-cycling PCR.
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR.
RAPD: Rapid amplified polymorphic DNA analysis.
Detection of fungal plant pathogen using PCR, Extraction of DNA from plant tissues,PCR amplification and detection of diagnostic amplicon
Polymerase chain reaction (PCR) is a technique in molecular biology used to
amplify (multiply) a single copy or a few copies of a piece of DNA, generating
thousands to millions of copies of that particular DNA sequence.
PCR is a revolutionary molecular biology technique used for enzymatically replicating DNA . This technique allows a small amount of DNA molecule to be amplified many times in an exponential manner . It is commonly used in medical and biological research labs for variety of tasks such as detection of hereditary disease , identification of genetic fingerprints diagnosis of infectious disease , cloning of genes and paternity testing .
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies . The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR.
There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95°C. For example, the half-life of Taq polymerase at 95°C is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100°C. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.
At 75°C, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute!
Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Direct PCR cloning methods include TA and GC cloning, as well as TOPO® Cloning, and enable direct cloning of PCR fragments. For example, the TA cloning approach takes advantage of the 3’ A overhang naturally added to products by Taq polymerase following PCR. The resulting sticky ends then enable recombination with DNA fragments containing 3’ T overhangs, such as linearized vectors.
During indirect PCR cloning, the PCR products are modified prior to recombination with other DNA sequences. For example, in restriction cloning, restriction sites are frequently introduced via PCR to enable restriction digestion and ligation with linearized vectors. PCR mutagenesis is a technique used to generate site-directed sequence changes such as base substitutions, inserts and deletions.
To insert a single point mutation via mutagenesis, for example, PCR primers are designed that contain the desired base change, usually in the middle of the primer sequence. PCR is then performed with the mutagenic primers and a high-fidelity DNA polymerase, which results in the incorporation of the desired mutation into the original sequence.Allele-specific PCR is used to detect sequence variations and ultimately determine the genotype of an organism.
For allele-specific PCR, primers are designed to flank the region of interest. The most common application of PCR is gene expression analysis
this ppt contain about pcr technique and its three process,primers in pcr,dna polymerase in pcr,melting temp of dna in pcr and applications of pcr technology
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.
PCR (polymerase chain reaction) and Extraction of DNA from fungal plant patho...AjayDesouza V
PCR, Polymerase chain reaction, types of PCR, Template DNA, DNA polymerase, Primers, Nucleotides (DNTPs or deoxynucleotide triphosphates ), Denaturation, Annealing, Extension, Types of PCR, Multiplex PCR.
Long-range PCR.
Single-cell PCR.
Fast-cycling PCR.
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR.
RAPD: Rapid amplified polymorphic DNA analysis.
Detection of fungal plant pathogen using PCR, Extraction of DNA from plant tissues,PCR amplification and detection of diagnostic amplicon
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
(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.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
1. PCR
The Polymerase Chain Reaction (PCR) provides an extremely sensitive
means of amplifying relatively large quantities of DNA
First described in 1985, Nobel Prize for Kary Mullis in 1993
The technique was made possible by the discovery of Taq polymerase, the
DNA polymerase that is used by the bacterium Thermus aquaticus that was
discovered in hot springs
The primary materials, or reagents, used in PCR are:
- DNA nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want to amplify
- Primers, single-stranded DNAs between 20 and 50 nucleotides
long (oligonucleotides) that are complementary to a short
region on either side of the template DNA
- DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA
PCR
DNA sequencing
Microarrays
Mass-spec
2. The Polymerase Chain Reaction (PCR) provides an extremely sensitive means
of amplifying relatively large quantities of DNA
First described in 1985, Nobel Prize for Kary Mullis in 1993
The technique was made possible by the discovery of Taq polymerase, the DNA
polymerase that is used by the bacterium Thermus aquaticus that was
discovered in hot springs
The primary materials, or reagents, used in PCR are:
- DNA nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want to amplify
- Primers, single-stranded DNAs between 20 and 50 nucleotides
long (oligonucleotides) that are complementary to a short
region on either side of the template DNA
- DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA
3. PCR
The cycling reactions :
There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is done on an
automated Thermo Cycler, which can heat and cool the reaction tubes in a very short time.
Denaturation at around 94°C :
During the denaturation, the double strand melts open to single stranded DNA, all enzymatic
reactions stop (for example the extension from a previous cycle).
Annealing at around 54°C :
Hydrogen bonds are constantly formed and broken between the single stranded primer and the
single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong
that the primer stays attached
Extension at around 72°C :
The bases (complementary to the template) are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added
complementary to the template)
PCR
DNA sequencing
Microarrays
Mass-spec
6. Every cycle results in a doubling of the number of strands
DNA present
After the first few cycles, most of the product DNA strands
made are the same length as the distance between the
primers
The result is a dramatic amplification of a the DNA that
exists between the primers. The amount of amplification is
2 raised to the n power; n represents the number of cycles
that are performed. After 20 cycles, this would give
approximately 1 million fold amplification. After 40 cycles
the amplification would be 1 x 1012
8. The most important consideration in PCR is contamination
Even the smallest contamination with DNA could affect amplification
For example, if a technician in a crime lab set up a test reaction (with
blood from the crime scene) after setting up a positive control reaction
(with blood from the suspect) cross contamination between the
samples could result in an erroneous incrimination, even if the
technician changed pipette tips between samples. A few blood cells
could volitilize in the pipette, stick to the plastic of the pipette, and
then get ejected into the test sample
Modern labs take account of this fact and devote tremendous effort to
avoiding cross-contamination
9. Optimizing PCR protocols
While PCR is a very powerful technique, often enough it is not possible to
achieve optimum results without optimizing the protocol
Critical PCR parameters:
- Concentration of DNA template, nucleotides, divalent cations
(especially Mg2+) and polymerase
- Error rate of the polymerase (Taq, Vent exo, Pfu)
- Primer design
PCR can be very tricky
PCR
DNA sequencing
Microarrays
Mass-spec
10. Primer design
General notes on primer design in PCR
Perhaps the most critical parameter for successful PCR is the design of primers
Primer selection
Critical variables are:
- primer length
- melting temperature (Tm)
- specificity
- complementary primer sequences
- G/C content
- 3’-end sequence
Primer length
- specificity and the temperature of annealing are at
least partly dependent on primer length
- oligonucleotides between 20 and 30 (50) bases are highly sequence specific
- primer length is proportional to annealing efficiency: in general, the longer the
primer, the more inefficient the annealing
- the primers should not be too short as specificity decreases
PCR
DNA sequencing
Microarrays
Mass-spec
14. DNA Sequencing
Snapshots of the
detection of the
fragments on the
sequencer
four-dye system single-dye system
PCR
DNA sequencing
Microarrays
Mass-spec
16. DNA Sequencing
The linear amplification of the gene in sequencing
PCR
DNA sequencing
Microarrays
Mass-spec
17. Massively parallel measurements of gene
expression: microarrays
• Defining the “transcriptome”
• The northern blot revisited
• Detecting expression of many genes: arrays
• A typical array experiment
• What to do with all this data?
Brown and Botstein (1999) “Exploring the new world
of the genome with DNA microarrays” Nature
Genetics 21, p. 33-37.
19. 1) Study all transcripts at same time
2) Transcript abundance usually correlates with level
of gene expression--much gene control is at level
of transcription
3) Changes in transcription patterns often occur as a
response to changing environment--this can be
detected with a microarray
20. Detection of mRNA transcripts
• Northern Blot -- immobilize mRNA on membrane,
detect specific sequence by hybridization with one
labeled probe--requires a separate blotting for
each probe
• DNA microarray -- immobilize many probes
(thousands) in an ordered array, hybridize (base
pair) with labelled mRNA or cDNA
21. Generating an array of probes
Identify open reading frames (orfs)
1) PCR each orf (several for each orf), attach (spot)
each PCR product to a solid support in a specific
order (pioneered by Pat Brown’s lab, Stanford)
2) Chemically synthesize orf-specific oligonucleotide
probes directly on microchip (Affymetrix)
22. http://derisilab.ucsf.edu/microarray/
(Derisi Lab at UCSF)
The chip defines
the genes you are
measuring
The hybridization
represents the
measurement
The RNA comes
from the cells and
conditions you are
interested in
23.
24. A print head for generating arrays of probes
Print head travels from DNA probe source
(microtiter plate) to solid support (treated glass
slide)
Small amount of DNA probe is put on a specific
spot at a specific location
Each spot (DNA probe sequence) has a specific
“address”
QuickTime™ and a
TIFF (Uncompressed) decom press or
are needed to s ee this picture.
QuickTime™ anda
TIFF ( Uncompressed) decompressor
are neededtosee this pictur e.
Print head
Printing needles
25.
26.
27. A yeast array experiment
vegetative sporulating
Isolate mRNA
Prepare fluorescently labeled
cDNA with two different-
colored fluors
hybridize read-out
28. Example microarray data
Green: mRNA more
abundant in vegetative
cells
Red: mRNA more
abundant in sporulating
cells
Yellow: equivalent mRNA
abundance in vegetative
and sporulating cells
29. Mass spectrometry for identifying proteins in a
mixture
From J.R. Yates 1998 “Mass spectrometry and the age of the proteome” J Mass
Spec. 33, p 1-19
Liquid chromatography and
tandem mass spectrometry
Software for processing data
30. Shotgun sequencing
Shotgun sequencing dispenses with the need for
mapping and so is much faster. It involves chopping
the DNA into fragments of size c. 2000 base pairs
(bps) and 10000 bps, sequencing the first and last 500
bps of each fragment. It then uses computer
algorithms to assemble the entire sequence from the
sequenced fragments.
31. Speed and accuracy of sequencing
Shotgun sequencing is much faster- it took a matter of
months to obtain a draft sequence of the fruit-fly,
Drosophila Melanogaster (135Mbps), when the
state-funded conventional sequencing effort had taken
several years to achieve a similar level of completion.
BUT assembly of pieces, in eg. the human (3x109 bps),
requires very powerful computers
AND repetitive DNA, which is common in eukaryotic
genomes, causes great difficulties in the assembly
process – may get it wrong.
32. Conclusions
Sequencing DNA involves:
Amplifying it by PCR or cloning
Chopping it up into manageable bits
Replicating it with fluorescently-tagged
dideoxynucleotides
Running the different length fragments on a gel and
reading this
Assembling the pieces (sequences of manageable bits).
Shotgun sequencing is faster than mapping-based
assembly methods, but can have accuracy
problems.
33. Sequence data is stored in online databases
Extracting useful information and patterns from such
data is part of bioinformatics and often employs
intelligent systems techniques.
