The analysis of all transcripts within a cell is of essential importance. Molecular biology provides many approaches to clone RNA transcripts into cDNA. Large cDNA collections are in the public domain to serve the research community. Today, however, new high-speed sequencing methods allow a much deeper view into transcriptomes than possible by classical cloning.
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
cDNA library construction using mRNA which are derived from DNA. cDNA is formed from the reverse transcription of single stranded mRNA. cDNA contains only the exons, it donot not contains introns. The mRNA consists of poly A tail in which the tRNA and rRNA donot contains poly A tail. A short oligo nucleotide of Poly T is used to isolate mRNA seperately thereby single stranded mRNA is then converted into cDNA by using reverse transcriptase enzyme.
Genome: The entire chromosomal genetic material of an organism.
Sequencing a genome: Determining the identity and order of nucleotides in the genetic material – usually DNA, sometimes RNA, of an organism.
DNA Libraries are collection of fragments of DNA cloned to a vector so that researchers can easily identify and isolate a particular gene of interest for future use.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
cDNA library construction using mRNA which are derived from DNA. cDNA is formed from the reverse transcription of single stranded mRNA. cDNA contains only the exons, it donot not contains introns. The mRNA consists of poly A tail in which the tRNA and rRNA donot contains poly A tail. A short oligo nucleotide of Poly T is used to isolate mRNA seperately thereby single stranded mRNA is then converted into cDNA by using reverse transcriptase enzyme.
Genome: The entire chromosomal genetic material of an organism.
Sequencing a genome: Determining the identity and order of nucleotides in the genetic material – usually DNA, sometimes RNA, of an organism.
DNA Libraries are collection of fragments of DNA cloned to a vector so that researchers can easily identify and isolate a particular gene of interest for future use.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
Genome editing with the CRISPR-Cas9 system has become one of the major tools in modern biotechnology. This slide share discusses the fundamentals in a simple, easy to understand format.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
High throughput next generation sequencing and robust transcriptome analysis help with gene expression profiling, gene annotation or discovery of non-coding RNA.
In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
Genome editing with the CRISPR-Cas9 system has become one of the major tools in modern biotechnology. This slide share discusses the fundamentals in a simple, easy to understand format.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
High throughput next generation sequencing and robust transcriptome analysis help with gene expression profiling, gene annotation or discovery of non-coding RNA.
In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
DNA cloning is a technique for reproducing DNA fragments.
It can be achieved by two different approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
a vector is required to carry the DNA fragment of interest into the host cell.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes.
This chapter provides an overview of DNA microarrays. Microarrays are a technology in which 1000’s of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events. We first cover the history of microarrays and the antecedent technologies that led to their development. We then discuss the methods of manufacture of microarrays and the most common biological applications. The chapter ends with a brief discussion of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
As a pioneer biotechnology company in the world, Creative Biogene can provide high quality cDNA libraries construction service to customers worldwide.
https://www.creative-biogene.com/Services/cDNA-Library-Construction-Service
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
The complete genome sequences of a number of organisms, including mammals, have recently become available because of rapid advances in DNA sequencing technology. Nevertheless, the analysis of transcripts still plays a crucial role in bridging the gap between the genome and the proteome, particularly in mammals. Therefore, as a method for the analysis of transcripts, cDNA library construction is crucial, even in the post-genome sequencing era.
https://www.creative-biogene.com/Services/cDNA-Library-Construction-Service
Creative Biogene’s goal is to provide you with the most affordable and high-quality cDNA libraries construction service to ensure your satisfaction in a timely and professional manner. https://www.creative-biogene.com/Services/cDNA-Library-Construction-Service
Genomic projects provided clone resources for coding mRNAs, but non-coding mRNAs are still missing for functional studies. Seeing the growing number of non-coding mRNAs, we hope the community will prepare better resources for studying human non-coding mRNAs.
Now a day's these technique is tremendously use for in lab by using foreign Dna to to producing insulin in bacteria , plant with high yielding capacity by using Gene from another species
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
2. Classical View on the Utilization of Genomic Information
Transcript Start Site Nucleus
Promoter “Gene”
Genomic DNA
(storage of information)
Transcription Factors
Transcription by RNA polymerase II
AAAAA Coding mRNA
Cap
(transport of information)
(7-methylguanosine cap or m7G cap)
Translation at ribosome
Protein
Cytoplasm
(tools to operate “functions”)
Developed in the 50th and 60th of last century. 2
3. The Classical View Has Been Challenged by new Developments
Discovery/Project Importance Year
Discovery of reverse DNA can be synthesized from RNA 1969
transcriptases templates
Discovery of ligase and Establishing DNA recombination, 1960s and 70s
restriction DNA cloning, and preparation of
endonucleases DNA libraries
DNA sequencing Chain-termination method 1975
(“Sanger Sequencing”)
Human Genome Project Move to sequencing entire genomes 1990 to 2003
Expressed sequence tags First attempt to gene discovery 1991
(ESTs) and expression profiling
IMAGE Project Program to create cDNA collections 1993 to 2007
from key organisms
ENCODE Project Functional elements in human Since 2003
genome
3
4. Topics of the Presentation
Approaches to cDNA cloning
Special topics related to cDNA cloning
Large-scale cDNA cloning projects
Small RNA (sRNA) cloning
Tag-based approaches
Next-Generation Sequencing
Where do we go from here?
4
5. Approaches to cDNA cloning
AAAAA 3’ Capped and polyadenylated mRNA
5’ Cap
Cap mRNA A A A A A… 1st Strand cDNA synthesis:
TTTTT Commonly oligo(dT) priming
mRNA
Prime 2nd strand cDNA synthesis:
Adaptor
cDNA 5’-Linker ligation or tailing reaction
2nd Strand synthesis
Adaptor cDNA
(Option to make PCR)
Digestion with cloning enzyme(s):
cDNA Methylation can protect against internal
cleavage within cDNA
Ligation into phage or plasmid vector:
PlPasmi
Plasmid
d
(Plasmid with cDNA insert may be
excised from phage vector)
Phage
5
6. Special Topics Related to cDNA Cloning
Synthesis of very long cDNAs (>10.000 bp, not further discussed)
Full-length cDNA cloning (important to obtain functional cDNAs)
Normalization (key to gene discovery in large-scale projects)
Cloning vectors and applications (not further discussed)
Subtractive cloning (not further discussed)
Expression cloning (not further discussed)
Addressing splicing (left out of large-scale projects)
Ref.: Harbers M: The current status of cDNA cloning, Genomics. 2008 Mar;91(3):232-42.
6
7. Use of cDNA Libraries
Isolation of individual target genes
in Research Laboratories
Transcriptome Analysis and Genome Projects
Large-scale random clone picking
End-sequencing to build transcript catalogs
Full-length sequencing of selected clones
Creation of sequence data bases
Creation of cDNA collections
Ref.: Carninci P et al.: Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.
Genome Res. 2003 Jun;13(6B):1273-89. 7
8. Benefits of Large-Scale cDNA Cloning Projects
Improved cDNA Cloning Technology
SNP Analysis:
Proteomics:
Sequence Data Location in Promoter or
Functional Studies on
Exon
Proteins
Clone Collections Functional Studies
Gene Regulation: Genomics:
Promoter Identification Gene Discovery
Expression Profiling Mapping
RNAi Noncoding RNA
Knock down Sense-antisense Pairs
Public sequence databases and clone collections are essential tools for research!
8
9. The mRNA Pool of a Cell
10,000 t0 20,000 transcripts
<20% of mRNA
5 t0 10 transcripts
up to 20% of mRNA
500 t0 2,000 transcripts
40 to 60 % of mRNA
(Old numbers estimated from
reassociation and hybridization studies)
Discovery of rarely expressed genes is a difficult task!
9
10. Normalization of cDNA Libraries
During a Normalization Step a cDNA pool is hybridized against an aliquot of the
original mRNA sample or the same cDNA pool. Due to concentration dependent
hybridization kinetics the number clones representing highly expressed genes will
be reduced yielding in a more equal distribution of different cDNAs in the library.
Without Normalization With Normalization Combine Normalization and
/Subtraction /Subtraction
Subtraction for higher Gene
/Hind III /Hind III
Discovery
9.4 kbp 9.4 kbp
6.6 kbp 6.6 kbp
Number of non-redundand clones
4.4 kbp 4.4 kbp
2.2 kbp 2.2 kbp Driver 2
2.0 kbp 2.0 kbp Lib. 4 +
Driver 2
Driver 1
Lib. 3 +
Driver 1
Lib. 2 No Driver
0.5 kbp 0.5 kbp
Lib. 1
: Highly expressed genes Example: Pancreas cDNA
Number of Libraries
10
11. Full-Length cDNA Cloning
“Cap Trapper” Method “Oligo Capping” Method
Cap P P P mRNA A A A A A…
Cap mRNA A A A A A… P mRNA A A A A A…
TTTTT
Phosphatase
Chemical reaction
Cap P P P mRNA A A A A A…
Biotin Cap mRNA mRNA A A A A A…
A A A A A…
cDNA TTTTT Pyrophosphatase
RNase I digestion P mRNA A A A A A…
mRNA A A A A A…
Biotin Cap mRNA A A A A A…
cDNA TTTTT RNA Ligase
Adaptor mRNA A A A A A…
Recovery on beads TTTTT
Biotin Cap mRNA
Beads A A A A A…
cDNA TTTTT Adaptor mRNA A A A A A…
cDNA TTTTT
Adaptor Primer
cDNA
cDNA
Key Steps: Key Steps:
Biotinylation of Cap structure and RNase I Treatment Replacement of Cap structure by RNA oligonucleotide
11
12. Examples for Large-Scale cDNA Cloning Projects
Targeting at the cloning and full-length sequencing of “one representative” cDNA clone for
each gene. This reduces cost, but it entirely ignores splicing events.
Project Organisms URL
IMAGE Consortium Human, mouse, rat, zebrafish, fugu, http://image.llnl.gov/
Xenopus (X. laevis and X. tropicalis),
cow, and primate
Mammalian Gene Human, mouse, rat, cow, others http://mgc.nci.nih.gov/
Collection (MGC)
Tokyo University Human http://cdna.hgc.jp/
RIKEN FANTOM Mouse http://fantom3.gsc.riken.go.jp/
Rice full-length cDNA Rice http://cdna01.dna.affrc.go.jp/cDNA/
Consortium
RIKEN Arabidopsis Arabidopsis http://www.brc.riken.jp/lab/epd/Eng/
news/071015.shtml
ORF Consortium Human (some mouse clones) http://www.orfeomecollaboration.org
12
13. Pre-mRNA is Spliced into mRNA
Large-scale cloning projects do not cover splice variants.
But maybe 75% of all signal transducers are regulated by splicing! 13
14. Capturing alternatively Spliced Exons in mRNA
Sense strand Antisense strand
Sample 1 Sample 2
Cut double-stranded regions
Capture single-stranded regions
Ref.: Watahiki A et al.: Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.
Nature Methods 2004 Dec 1(3): 233-9.
14
15. The Discovery of small RNAs
Classical cloning protocols removed all cDNA fragments of less than
500 bp (avoid linker contamination, cutoff of cloning vectors).
Proteins of less than 100 amino acids were commonly not annotated.
However, small RNAs have important functions!
Small RNAs are non-coding RNAs (ncRNAs) often derived from maturation
processes in the cell that include digestion steps by RNases.
Most prominent example: microRNAs (miRNA) have reverse complement
sequences to other mRNA transcripts. They are around 21-23 base pairs long
after maturation and can alter the expression/translation of one or several
target genes through RNA interference.
And we are still finding many more new RNA species!
Ref.: Kawaji H, Hayashizaki Y. Exploration of small RNAs. PLoS Genet. 2008 Jan;4(1):e22.
15
16. Small RNA (sRNA) Cloning
5’ P OH 3’ Short RNA
Modify 3’ end:
P CCCCCCCCC P
C-Tailing or adaptor ligation
Modify 5’ end:
CCCCCCCCC
Here by adaptor ligation
CCCCCCCCC
GGGGGGGG 1st Strand cDNA synthesis
CCCCCCCCC
GGGGGGGG 2nd Strand synthesis and PCR
Sequence analysis:
PlPasmi Direct sequencing of DNA fragments
Plasmid
d
(Option to ligate into plasmid vector)
Key Steps:
Modification of 5’ and 3’ end of RNA for PCR amplification. Selection by size range. Commonly only sequenced.
No cloning needed as short cDNAs can be chemically synthesized.
16
17. Tag-Based Approaches
Gene discovery cannot be done by standard methods used in
expression profiling such as microarray or PCR.
Unsupervised approaches are needed for gene discovery that do
not require sequence information for probe design.
First approach to gene discovery was sequencing of 3’ ends of cDNA
clones (EST sequencing). Requires one read per clone.
Gene identification does not require sequences of 500 to 800 bp,
but much shorter sequences of some 20 bp or less are sufficient.
Use long sequencing reads to cover many short fragments by one run.
New protocols to isolated short fragments from RNA.
Tag-based approaches in expression profiling and gene discovery.
Ref.: Harbers M and Carninci P: Tag-based approaches for transcriptome research and genome annotation.
Nature Methods 2005 Jul 2(7): 495-502.
17
18. Tag-Based Approaches
Paired-end Tags or PETs
5’ end 3’ end
Anchoring enzyme sites
Cap selection Remove poly(A)
Cap mRNA AAAAA
CAGE SAGE SAGE 3’ SAGE
5’ SAGE (5’ related) (3’ related)
MPSS
DGE
RNA-Seq
or other shotgun approaches
18
19. Serial Analysis Gene Expression (SAGE)
(Digital Gene Expression (DGE))
mRNA A A A A A… 1st Strand cDNA Synthesis with biotinylated primer
TTTTTT Biotin (Commonly starting from mRNA.)
cDNA
Biotin Beads Preparation of double-stranded cDNA and digestion with anchoring enzyme
Adaptor cDNA
Biotin Beads Adaptor Ligation and digestion with Mme I (20 bp) or EcoP15I (27 bp)
Adaptor Adaptor Formation of “Di-Tags”
(Di-Tags can be used for direct sequencing (DGE).)
Concatenation and cloning into plasmid vector
(Classic sequencing of concatemers.)
Very well established and rich reference/annotation information.
Digital expression profiling by “tag counting”.
Ref.: Velculescu VE et al. Serial analysis of gene expression. Science. 1995 Oct 20;270(5235):368-9, 371.
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20. Cap Analysis Gene Expression (CAGE)
5’ CAP mRNA AAAAA 3’ Commonly starting from 50g total RNA.
1st Strand cDNA Synthesis
(Covering poly(A-) mRNA and long mRNA.)
CAP mRNA AAAAA
cDNA NNNNNN
5’-End Selection on Beads by Cap Trapper
(Less bias due to chemical modification of Cap.)
Beads CAP mRNA AAAAA
cDNA NNNNNN
Adaptor Ligation and 2nd Strand Synthesis
Adaptor I
cDNA NNNNNN
Digestion with Mme I (20 bp) or EcoP15I (27 bp)
Adaptor I cDNA
Isolation of CAGE TAGs
Adaptor I TAG
3’-End Adaptor Ligation
Adaptor I TAG Adaptor II Preferably used for direct sequencing (>4,000,000 tags per run).
Ref.: Kodzius R et al.: Cap analysis of gene expression: transcription start site mapping and expression profiling.
Nature Methods 2006 Mar 3(3): 211-222. 20
21. Cap Analysis Gene Expression (CAGE)
Signal 1 Signal 2 Signal 3 CAP mRNA A A A A A
TSS
Genome TF1 TF2 TF3 Exon 1 2 3 4 5
Tiling Array/RNA-Seq
Array/RNA-
Microarray
TF CAGE Tags SAGE
ChIP RACE
CAGE tags experimentally link transcripts to their promoters.
CAGE tags integrate information based on genome annotations.
CAGE tags can be linked to whole genome tiling arrays and RNA-Seq data.
CAGE tags can be linked to Chromatin IP/ChIP-Seq data.
CAGE tags correlate with open chromatin.
CAGE tags provide primer information for cloning new transcripts.
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22. Classical DNA Sequencing by Chain-Termination Method
dNTP/ddNTP Mix
G C G
A T G
T
C C
A A A G C T
Primer T A
A C C A
DNA Template T G G T T G C T G C C A A T G T
One reaction per nucleotide
DNA Polymerase
A T G C T G G T T G C T G C C A A T G T
T G G T T G C T G C C A
T G G T T G
T G G T T G C T G C
Capillary Sequencer Analyze fragments DNA fragments from
by gel electrophoresis Primer extension reactions
Over 30 years the most important method in molecular biology.
Challenged by emerging new sequencing technologies: Next-Generation Sequencing.
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23. Next-Generation Sequencing
Driven by the “$1000 genome” different companies are on the move to provide new sequencing
technologies based on “sequencing by synthesis” or “ligation-based sequencing”. Other approaches
may use hybridization methods or physical means in the future.
Platform Mb per run/read length Method
Roche 454 Sequencing 100 Mb/250 bp/7h per run Emulsion PCR and Pyrosequencing
Illumina (Solexa) 1300 Mb/32-40bp/4 days per run Bridge PCR and sequencing-by-
synthesis
ABI SOLiD 3000 Mb/35 bp/5 days per run Emulsion PCR and ligation-based
sequencing
Helicos 25 to 90 Mb per h/up to 55 bp Single-molecule detection
Ref.: Mardis ER. The impact of next-generation sequencing technology on genetics.
Trends Genet. 2008 Mar;24(3):133-41. Epub 2008 Feb 11.
von Bubnoff A. Next-generation sequencing: the race is on. Cell. 2008 Mar 7;132(5):721-3.
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24. Example for Ligation-Based Sequencing: ABI SOLID System
DNA fragments having Project specific data analysis:
adaptor sequences: Mapping to genome
Genomic DNA Reference information
Tag Sequencing
Images are the courtesy of ABI and were kindly provided by ABI Japan.
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25. Example for Ligation-Based Sequencing: ABI SOLID System
Images are the courtesy of ABI and were kindly provided by ABI Japan. 25
26. Example for Ligation-Based Sequencing: ABI SOLID System
Images are the courtesy of ABI and were kindly provided by ABI Japan. 26
27. Example for Sequencing-by-Synthesis: Illumina 1G System
DNA per run Addition of Add to flow Preparation
0.1 ~1µg 2 adaptors cell of clusters
Images are the courtesy of Illumina and were kindly provided by Illumina Japan. 27
28. Example for Sequencing-by-Synthesis: Illumina 1G System
3’ 5’
Cycle 1
A Addition of the sequence reagent
T
C G One base extension reaction
C Removal of non-incorporated bases
G C
G Detect fluorescence signal
T A
A C T Removal of the fluorescence label
G
C Cycle 2
T C
C
C Repetition of the above reactions
C A G
T
A Cycle 3, 4, 5…..
T C
A
G C
Repetition of the above reaction
A
G
T
A G T
T G
T
5’ Images are the courtesy of Illumina and were kindly provided by Illumina Japan. 28
29. Example for Sequencing-by-Synthesis: Illumina 1G System
40,000,000 clusters on a flow cell
20um
100um
Images are the courtesy of Illumina and were kindly provided by Illumina Japan. 29
30. Where do we go from here?
Next-Generation Sequencing will push genome sequencing field for
re-sequencing and de novo sequencing (“1000 Genome Project”).
Metagenomics (Environmental Genomics, Ecogenomics, or
Community Genomics): Direct analysis of genetic materials obtained
from environmental samples.
Expression profiling: SAGE (DGE), CAGE, PET, RNA-Seq.
Analytical applications to identify functional regions/elements in
genomes: ChIP-Seq, open chromatin, SNPs, splicing, others to come .
Analytical applications in mutation screens.
Analytical applications for detection of infectious agents.
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31. Transcriptome Analysis: The Dominance of noncoding RNA
Genome sequencing and annotation did not tell us about the real
extent of gene expression!
Tiling array experiments and deep sequencing by next-generation
sequencing methods indicates that >90% of the genome is expressed.
Maybe 40 to 50% of the mRNA is not polyadenylated, and we did not
analyze it yet.
Most of the transcripts are potentially noncoding RNAs having
unknown (regulatory ?) functions.
The definition of a “gene” may no longer hold with many different
transcripts derived from same loci.
We do not understand the “hidden layers” regulating the utilization of
genomic information.
Ref.: Mattick, J.S. "Challenging the dogma: The hidden layer of non-protein-coding RNAs on complex organisms"
Bioessays. (2003) 25, 930-939.
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32. Example for RNA-Seq in Yeast Saccharomyces pombe (fission yeast)
Illumina 1G sequencer; average read length 39.1 base, fragments from poly(A) mRNA
> 23 mil reads (~60 genome length) proliferating cells.
> 99 mil reads (~ 190 genome length) from five different stages.
Covering ~94% nuclear and > 99% of mitochondrial genome.
Confirmed expression from intergenic regions by RT-PCR.
Control experiments using whole genome tiling arrays (25 mer/20 nt intervals)
confirmed identification novel transcripts (26 out of 453 may encode short
proteins).
Recent publications on the use of RNA-Seq include S. pombe, S. cerevisiae, Arabidopsis,
mouse tissues, mouse stem cells, and HeLa S3.
Ref.: Wilhelm BT, Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution.
Nature. 2008 Jun 26;453(7199):1239-43. Epub 2008 May 18.
Graveley BR. Molecular biology: power sequencing. Nature. 2008 Jun 26;453(7199):1197-8.
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33. Examples for Genome Size (haploid)
Genome Length in bp Estimated gene number
Phi-X 174 5,386 10
Human mitochondrion 16,569 37
E. coli 4,639,221 4,377
Saccharomyces cerevisiae 12,495,682 5,770
Caenorhabditis elegans 100,258,171 19,427
Arabidopsis thaliana 115,409,949 ~28,000
Drosophila melanogaster 122,653,977 13,379
Humans 3.3 x 109 ~20,500
Amphibians 109–1011 ?
Values taken from: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GenomeSizes.html out of July 2007
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34. Where are our limitations?
Mammalian genome size and transcriptome complexity:
Enrichment of fragments e.g. using microarrays,
Normalization and longer reads required.
Thus far uneven representation requires use of more than one method.
Requirements for starting materials (target is to analyze single cells).
No unified cDNA library method: using different methods depending on RNA length.
Very large data files and lack of computational analysis tools.
What is transcriptional noise?
Research dominated by “detection” rather than “functional analysis”.
Ref.: Struhl K. Transcriptional noise and the fidelity of initiation by RNA polymerase II.
Nat Struct Mol Biol. 2007 Feb;14(2):103-5.
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35. Present Strategies for Transcriptome Analysis
Interest has shifted to next-generation sequencing to profile transcriptional
activities.
We cannot predict ends of transcripts, and therefore tag-based approaches
to indentify start sites and termination sites are needed.
Identification of transcription start sites in combination with other
information is driving “gene networks studies” and “system biology”.
RNA-Seq provides new means for the identification of splice sites and
expressed mutations.
We do not clone all those new transcripts, but there will be a need to get
resources for functional analysis of new transcripts.
We are more than ever falling short on the functional analysis of new transcripts.
Thus far we have not even analyzed all coding transcripts!
It is an exciting time to work on transcriptome analysis offering many challenges and rewards!
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36. Contact:
Dr. Matthias Harbers
DNAFORM Inc.
Leading Venture Plaza-2, 75-1, Ono-cho
Tsurumi-ku, Yokohama City, Kanagawa, 230-0046
Japan
E-mail: matthias.harbers@dnaform.jp
Phone: +81-(0)45-510-0607
FAX: +81-(0) 45-510-0608
URL: http://www.dnaform.jp
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