mb206-jan09
DESCRIPTION
MB206-Jan09. Project. Samples : Plant (A) . Objective: Isolate 100 ESTs from Plant (A). RNA Extraction. Extract RNA from sample (A) – method depend on sample. Check previous note. Check the quality and quantity of the RNA. Isolate mRNA from the RNA (using kits) - PowerPoint PPT PresentationTRANSCRIPT
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MB206-Jan09Project
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Samples : Plant (A)
Objective:
Isolate 100 ESTs from Plant (A)
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RNA Extraction
Extract RNA from sample (A) – method depend on sample. Check previous note.Check the quality and quantity of the RNA.Isolate mRNA from the RNA (using kits)Check the quality and quantity of the mRNA. Then?
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mRNA isolation
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Most eukaryotic mRNAs are polyadenylated at their 3’ ends
• oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.
AAAAAAAAAAn5’ cap
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Methods:
Analysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels
Check the mRNA integrity
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Make sure that the mRNA is not degraded.
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Cloning the particular mRNAs
Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gels
Enrichment: carried out by hybridizationExample: clone the hormone induced mRNAs (substrated cDNA library)
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Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library.
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Types of Libraries
(5’UTR) (3’UTR)Angelia 09
8
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cDNA Libraries
polyA
polyA
polyA
Genomic DNA
Genomic DNA
mRNAReverse
transcribe
(and more)
Digest DNA fragments
cDNA
Clone in vector
cDNA library
Genomic DNA library
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Choosing a VectorUsually you select a vector (plasmid, λ, other) depending on how big you want your DNA fragments to be & the capacity of the vector.
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Lambda Library Lodish, et al. Fig 7-12
Plasmid Library Lodish, et al. Fig 7-1
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Plasmid !!!
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cDNA libraries
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1.No cDNA library was made from prokaryotic mRNA.
• Prokaryotic mRNA is very unstable
• Genomic libraries of prokaryotes are easier to make and contain all the genome sequences.
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cDNA libraries
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2.cDNA libraries are very useful for eukaryotic gene analysis • Condensed protein encoded gene libraries, have
much less junk sequences.• cDNAs have no introns genes can be
expressed in E. coli directly• Are very useful to identify new genes• Tissue or cell type specific (differential expression
of genes)
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Synthesis of cDNA :
First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1.1)
Second strand synthesis: best way of making full-length cDNA is to ‘tail’ the 3’-end of the first strand and then use a complementary primer to make the second.
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5’ mRNA AAAAA-3’ HO-TTTTTP-5’
5’
Reverse transcriptaseFour dNTPs
AAAAA-3’TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
AAAAA-3’TTTTTP-5’
TTTTTP-5’
3’
3’-CCCCCCC
Terminal transferasedCTP
Alkali (hydrolyaes RNA)Purify DNA oligo(dG)
Klenow polymerase or reverseTranscriotase Four dNTPs
5’-pGGGG-OH
5’
3’-CCCCCCC
5’-pGGGG3’-CCCCCCC TTTTTP-5’
-3’
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5’-pGGGG3’-CCCCCCC
HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC
5’-pAATTCGGGGGG
TTTTTGGCTTAAGCC-OH CCGAATTCGG-3’
3’-CCCC
3’-CCCCCCC
3’-CCC 5’-pGGGG
5’-pGGGG
TTTTTp-5’ -3’
TTTTTp-5’
TTTTTp-5’
-3’
-3’
TTTTTGGCTTAAp-5’
HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH
CCG-3’
Duplex cDNA
Single strand-specific nuclease
Klenow polymerase
treat with E.coRI methylase
Add E.colRI linkers using T4 DNA ligase
E.colRI digestion
Ligate to vector and transfom
Fig2.1 Second strand synthesis 19
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Treatment of cDNA endsBlunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.
The process :Move protruding 3’-ends (strand-special nuclease)
Fill in missing 3’ nucleotide (klenow fragment of DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers(T4 DNA ligase)
Restriction enzyme digestion (E.coRI )
Tailing with terminal transferase or using adaptor molecules
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Ligation to vector Any vectors with an EcoRI site would suitablefor cloning the cDNA.
The process : Dephosphorylate the vector with alkalinephosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)21
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Screening
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Screening
The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .
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Plate the cDNA library on LB agar plates-It need the help of host.-The detail can refer any cDNA library construction kits.
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Verification
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Verification
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Expressed Sequence Tag (EST)
Messenger RNA (mRNA) sequences in the cell represent copies from expressed genes.
RNA cannot be cloned directlyreverse transcribed to double-stranded cDNA
The resultant cDNA is cloned to make libraries representing a set of transcribed genes of the original cell, tissue or organism.
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Characteristics of EST sequences
Nagaraj, S. H. et al. Brief Bioinform 2007 8:6-21; doi:10.1093/bib/bbl015
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Sequencing
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DNA sequencing by the Sanger method
The standard DNA sequencing technique is the Sanger method, named for its developer, Frederick Sanger, who shared the 1980 Nobel Prize in Chemistry. This method begins with the use of special enzymes to synthesize fragments of DNA that terminate when a selected base appears in the stretch of DNA being sequenced. These fragments are then sorted according to size by placing them in a slab of polymeric gel and applying an electric field -- a technique called electrophoresis. Because ofDNA's negative charge, the fragments move across the gel toward the positive electrode. The shorter the fragment, the faster itmoves. Typically, each of the terminating bases within the collection of fragments is tagged with a radioactive probe foridentification.
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DNA sequencing exampleProblem Statement: Consider the following DNA sequence (from firefly luciferase). Draw the sequencing gel pattern that forms as a result of sequencing the following template DNA with ddNTP as the capper.
atgaccatgattacg...
Solution:
Given DNA template: 5'-atgaccatgattacg...-3' DNA synthesized: 3'-tactggtactaatgc...-5'
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DNA sequencing example Given DNA template: 5'-atgaccatgattacg...-3' DNA synthesized: 3'-tactggtactaatgc...-5'Gel pattern: +-------------------------+
lane ddATP | W | | || |lane ddTTP | W | | | | | |lane ddCTP | W | | | |lane ddGTP | W || | |
+-------------------------+ Electric Field + Decreasing size
where "W" indicates the well position, and "|" denotes the DNA bands on the sequencing gel.
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A sequencing gel
This picture is a radiograph. The dark color of the lines isproportional to the radioactivity from 32P labeled adenonsinein the transcribed DNA sample.
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Reading a sequencing gelYou begin at the right, which are the smallest DNA fragments. The sequence that you read will be in the 5'-3' direction. This sequence will be exactly the same as the RNA that would be generated to encode a protein. The difference is that the T bases in DNA will be replaced by U residues. As an example,in the problem given, the smallest DNA fragment on the sequencing gel is in the C lane, so the first base is a C. The next largest band is in the G lane, so the DNA fragment of length 2 ends in G. Therefore the sequence of the first two bases is CG. The sequence of the first 30 or so bases of the DNA are: CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC.... When this is made as RNA, its sequence would be: CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC.... Note that the information content is the same, only the T's have been replaced by U's!.
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The codon table 5’-Base Middle Base 3’-Base U(=T) C A G U(=T) Phe Ser Tyr Cys U(=T) Phe Ser Tyr Cys C Leu Ser Term Term A Leu Ser Term Trp G C Leu Pro His Arg U(=T) Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G A Ile Thr Asn Ser U(=T) Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp Gly U(=T) Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G
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Translating the DNA sequence
The order of amino acids in any protein is specificed by the order of nucleotide bases in the DNA. Each amino acid is coded by the particular sequence of three bases. To convert a DNA sequence First, find the starting codon. The starting codon is always the codon for the amino acid methionine. This codon is AUG in the RNA (or ATG in the DNA): GCGCGGGUCCGGGCAUGAAGCUGGGCCGGGCCGUGC.... MetIn this particular example the next codon is AAG. The first base (5'end) is A, so that selects the 3rd major row of the table. The second base (middle base) is A, so that selects the 3rd column of the table. The last base of the codon is G, selecting the last line in the block of four.
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Translating the DNA sequence
This entry AAG in the table is Lysine (Lys). Therefore the second amino acid is Lysine.
The first few residues, and their DNA sequence, are as follows (color coded to indicate the correct location in the codon table): Met Lys Leu Gly Arg … ...
AUG AAG CUG GGC CGG GCC GUG C..
This procedure is exactly what cells do when they synthesizeproteins based on the mRNA sequence. The process of translationin cells occurs in a large complex called the ribosome.
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Automated procedure for DNA sequencing
A computer read-out of the gel generates a “false color” imagewhere each color corresponds to a base. Then the intensities aretranslated into peaks that represent the sequence.
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High-throughput seqeuncing:Capillary electrophoresis
The human genome project has spurred an effort to develop faster, higher throughput, and less expensive technologies for DNA sequencing. Capillary electrophoresis (CE) separation has many advantages over slab gel separations. CE separations are faster and are capable of producing greater resolution. CE instruments can use tens and even hundreds of capillaries simultaneously. The figure show a simpleCE setup where the fluorescently-labeled DNA is detected as itexits the capillary.
Laser
PMT
Focusing lens
Sheath flow cuvette
Sheath flow
Collection LenscCollection Lensc
Beam block
filter
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DNA sequencing.Dideoxy analogs of normal nucleotide triphosphates (ddNTP) cause premature termination of a growing chain of nucleotides.
ACAGTCGATTGACAddGACAGTCddGACAGTCGATTddG
Fragments are separated according to their sizes in gel electrophoresis. The lengths show the positions of “G” in the original DNA sequence.
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Nucleotides and phosphodiester bond.
Phosphodiester bond
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Genomic sequencing.Individual chromosomes are broken into 100kb random fragments.This library of fragments is screened to find overlapping fragments – contigs.Unique overlapping clones are chosen for sequencing.Put together overlapping sequenced clones using computer programs.
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Sequencing cDNA libraries.
mRNA is pooled from the tissues which express genes.
cDNA libraries are prepared by copying of mRNA with reverse transcriptase.
Expressed Sequence Tags (EST) – partial sequences of expressed genes.
Comparing translated ESTs to annotated proteins – annotation of genes.
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Gene prediction.Gene – DNA sequence encoding
protein, rRNA, tRNA …
Gene concept is complicated:- Introns/exons- Alternative splicing- Genes-in-genes- Multisubunit proteins
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Gene structure.ATG TER
-35 -10
Promoter sequences
ATG – start codon; TER (TAA, TAG,TGA) – termination codons
Gene
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Codon usage tables.- Each amino acid can be encoded by several codons.
- Each organism has characteristic pattern of codon usage.
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Problems arising in gene prediction.
Distinguishing pseudogenes (not working former genes) from genes.Exon/intron structure in eukaryotes, exon flanking regions – not very well conserved.Exon can be shuffled alternatively – alternative splicing.Genes can overlap each other and occur on different strands of DNA.
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Gene identificationHomology-based gene prediction• Similarity Searches (e.g. BLAST, BLAT)• ESTsAb initio gene prediction• Prokaryotes
ORF identification• Eukaryotes
Promoter predictionPolyA-signal predictionSplice site, start/stop-codon predictions
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Prokaryotic genes – searching for ORFs.
- Small genomes have high gene density Haemophilus influenza – 85% genic - No introns- Operons One transcript, many genes- Open reading frames (ORF) – contiguous set of codons, start with Met-codon,
ends with stop codon.
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Example of ORFs.There are six possible ORFs in each sequence for both directions of transcription.
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Confirming gene location using EST libraries.
Expressed Sequence Tags (ESTs) – sequenced short segments of cDNA. They are organized in the database “UniGene”.
If region matches ESTs with high statistical significance, then it is a gene or pseudogene.