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)Check the quality and quantity of the mRNA. Then?

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

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.

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.

Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library.

Types of Libraries

(5’UTR) (3’UTR)

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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 !!!Plasmid !!!

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.

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)

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. Angelia 09 17

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

Treatment of cDNA ends

Blunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.

Blunt 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)

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Screening

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.

Verification

Verification

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.

Characteristics of EST sequences

Nagaraj, S. H. et al. Brief Bioinform 2007 8:6-21; doi:10.1093/bib/bbl015

Sequencing

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.

DNA sequencing example

Problem 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'

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.

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.

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!.

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

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.

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.

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.

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

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.

Nucleotides and phosphodiester bond.

Phosphodiester bond

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.

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.

Gene prediction.

Gene – DNA sequence encoding protein, rRNA, tRNA …

Gene concept is complicated:- Introns/exons- Alternative splicing- Genes-in-genes- Multisubunit proteins

Gene structure.

ATG TER

-35 -10

Promoter sequences

ATG – start codon; TER (TAA, TAG,TGA) – termination codons

Gene

Codon usage tables.- Each amino acid can be encoded by several codons.

- Each organism has characteristic pattern of codon usage.

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.

Gene identificationHomology-based gene prediction• Similarity Searches (e.g. BLAST, BLAT)• ESTs

Ab initio gene prediction• Prokaryotes

ORF identification• Eukaryotes

Promoter predictionPolyA-signal predictionSplice site, start/stop-codon predictions

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.

Example of ORFs.There are six possible ORFs in each sequence for both directions of transcription.

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.