MB206-Jan09

1. Project MB206-Jan09

2. Samples : Plant (A) Objective: Isolate 100 ESTs from Plant (A)

4. 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?

5. Types of Libraries Angelia 09 (3’UTR) (5’UTR)

6. Lambda Library Lodish, et al. Fig 7-12 Plasmid Library Lodish, et al. Fig 7-1 Angelia 09

7. Plasmid !!!

9. Screening

10. 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 . • Plate the cDNA library on LB • agar plates • It need the help of host. • The detail can refer any cDNA library construction kits. Angelia 09

11. Sequencing

12. 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 of DNA's negative charge, the fragments move across the gel toward the positive electrode. The shorter the fragment, the faster it moves. Typically, each of the terminating bases within the collection of fragments is tagged with a radioactive probe for identification.

13. 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'

14. 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.

15. A sequencing gel This picture is a radiograph. The dark color of the lines is proportional to the radioactivity from 32P labeled adenonsine in the transcribed DNA sample.

16. Reading a sequencing gel You 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!.

17. The codon table

18. 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.... Met In 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.

19. 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 synthesize proteins based on the mRNA sequence. The process of translation in cells occurs in a large complex called the ribosome.

20. Automated procedure for DNA sequencing A computer read-out of the gel generates a “false color” image where each color corresponds to a base. Then the intensities are translated into peaks that represent the sequence.

21. 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 simple CE setup where the fluorescently-labeled DNA is detected as it exits the capillary. Sheath flow Laser Focusing lens Sheath flow cuvette Beam block Collection Lensc Collection Lensc PMT filter

22. DNA sequencing. • Dideoxy analogs of normal nucleotide triphosphates (ddNTP) cause premature termination of a growing chain of nucleotides. ACAGTCGATTG ACAddG ACAGTCddG ACAGTCGATTddG • Fragments are separated according to their sizes in gel electrophoresis. The lengths show the positions of “G” in the original DNA sequence.

23. Nucleotides and phosphodiester bond. Phosphodiester bond

24. 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.

25. 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.

26. Gene prediction. Gene – DNA sequence encoding protein, rRNA, tRNA … Gene concept is complicated: • Introns/exons • Alternative splicing • Genes-in-genes • Multisubunit proteins

27. Gene structure. ATG TER -35 -10 Promoter sequences Gene ATG – start codon; TER (TAA, TAG,TGA) – termination codons

28. Codon usage tables. • Each amino acid can be encoded by several codons. • Each organism has characteristic pattern of codon usage.

29. 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.

30. Gene identification • Homology-based gene prediction • Similarity Searches (e.g. BLAST, BLAT) • ESTs • Ab initio gene prediction • Prokaryotes • ORF identification • Eukaryotes • Promoter prediction • PolyA-signal prediction • Splice site, start/stop-codon predictions

31. 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.

32. What is Sequencing A lab technique used to find out the sequence of nucleotide bases in a DNA molecule or fragment. It is a deciphering of the exact order of base sequence in a nucleotide Sequence. Examples are dideoxy sequencing and maxam-gilbert sequencing.

33. Method of the Sequencing There are two methods of sequencing Maxam and Gilbert method (the manual or chemical sequencing ) And Sanger method using dideoxynucleotide (modern sequencing)

34. Sanger method is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice.