Dideoxy or Chain Termination MethodDetermining precise sequence of nucleotides in a DNA sample

DNA SEQUENCING

1974

Maxam/Gilbert (USA) (Chemical Cleavage protocol)

Sanger (England) (natural process of DNA replication)*

Phosphodiester bond cannot form with incoming nucleotides leading to termination of DNA synthesis

Primer

ADD:

Chromatogram: Coloured peaks corresponding to specific nucleotides in specific locations

Expressed Sequence Tags (ESTs)

ESTs are short DNA sequences (usually 200 to 500 nt) that are generated by sequencing either one or both ends of an expressed gene.

These small strands of DNA represent genes expressed in certain cells from different organisms. They are used as "tags" to fish a gene out of a portion of chromosomal DNA by matching base pairs.

Challenge of gene identification: - dependent upon genome size (number of introns/exons).

Isolating mRNA is key to identifying expressed genes. You now want to work with this mRNA sequence. However, mRNA is very unstable outside the cell.Therefore the mRNA is converted to cDNA (complementary DNA) which is muchmore stable outside the cell.

cDNA is synthesized by reverse transcribing the mRNA. Since cDNA is made directly from mRNA, it represents ONLY expressed DNA sequence (the gene!)

Synthesis of ESTs

                                        

REVERSE TRANSCRIPTION

Reverse Transcriptase

Reverse transcriptase is a common name for an enzyme that functions as a RNA-dependentDNA polymerase. They are encoded by retroviruses, where they copy the viral RNA genome into DNA prior to its integration into host cells.

Reverse transcriptases have 2 Main Functions:

1. DNA polymerase activity: Needs primer to initiate synthesis (oligodT)

2. RNase H activity: RNase H is a ribonuclease that degrades the RNA from RNA-DNA hybrids, such as are formed during reverse transcription of an RNA template.

All retroviruses have a reverse transcriptase, but the enzymes that are available commercially are derived from one of two retroviruses,

Moloney murine leukemia virus: a single polypeptide

*Avian myeloblastosis virus: composed of two peptide chains

Use T7 sequencing primer

Your plasmid will be sent for sequencing this week.I will analyze each EST sequence using the database prior to submissionSequence should be posted by Tues, Feb 6. Your assignment 1 is due Feb 19 giving you sufficient time to finish it

EcoRIHindIII

Until your sequence comes in, you may use the sample sequence posted on the course website to practice how to navigate your way around the NCBI website.

Results and Assignments: Results are handed in with the assignments (See course Website for details)

Although the assignments rely on group results, each student will hand in their own results and assignment.

I cannot help you with the assignments. Why? They are essentially your “tests” since there are no midterm exams

Results and Assignment 1 is the most time consuming so start the assignment as soon as your EST sequence is uploadedonto the course website. Meanwhile, I recommend you to do the following:

-Learn how to use and explore the Honeybee Databaseusing sample sequence -There is extensive on-line help on how to use this site-You must learn to do this independently

Page 1: Picture of your agarose gel from experiment 1- You must LABEL your lane very clearly. Include

scientific figure legend and comments about the gel.

Page 2: Print out of the ‘Mock Sequence Submission’

Pages 3 and 4: Analysis of the results (2 pages only)12 point font

1.5 line spacing

1 inch margins

Figures included in the 2 pages

This must be individual work; no collaboration.

You will use NCBI Website (http:/www.ncbi.nlm.nih.gov/) to do this assignment.

PURPOSE OF ASSIGNMENT 1: To test your ability to learn how to use an electronic database available for genetic research. (The Honeybee Genome Database)

Assignment 1 Format (Con’t)

-Incorporate answers to all questions into 2 pages. -Figures should be useful and kept to a minimal size.

6. To get full marks you will have to explore the web for at least 4 other important information about the gene that you have not been asked. For example, what do we know about the role of the gene in other organisms? This is just one example, you don’t have to use this.

Not a figure Not a figure Good figure

I will help you with Assignment 1 today.

What is NCBI?

The National Center for Biotechnology Information was established in 1988 as a national resource for molecular biology information. It creates public databases, conducts research in computational biology, develops software tools for analyzing genome data. The goal is for this information to ultimately lead to a better understanding of molecular processes

affecting human health and disease

EST Sequence Submission and Analysis

1. Mock Sequence Submission To GenBank

GenBank is a large database kept by the US government which includes nucleotide sequences submitted by scientists worldwide.

Using Sequin to submit data to GenBank: -software developed by NCBI to submit and update sequencesto GenBank, EMBL, DDBJ.-goal of sequin is to convert raw sequence data to an assembled record that can be viewed, edited and submitted to databases.-data is normally submitted in FASTA format.

2. Analysis of EST sequence

BLAST (Basic Local Alignment Search Tool) compares a querynucleotide (or protein) sequence to a database of known sequences in order to determine similarity to previously published sequences. If the sequence has not yet been published, it provides insight into the function of the DNA or protein.

BLAST Search

blastn compares your query nucleotide sequence with database nucleotide sequences.

blastx translates your query sequence into amino acids and then compares the protein sequences with protein databases.

FASTA is a DNA and Protein Sequence Alignment software package first described in 1988. It stems from the original FASTP (protein vs All).

The FASTA program is a program which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched.

The FASTA program follows a largely heuristic method which contributes to the high speed of its execution.

The format allows for sequence names and comments to precede the sequences. Example:>gi|5524211|gb|AAD44166.1| cytochrome b [Elephas maximus maximus] LCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGTNLVEWIWGGFSVDKATLNRFFAFHFILPFTMVALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLGLLILILLLLLLALLSPDMLGDPDNHMPADPLNTPLHIKPEWYFLFAYAILRSVPNKLGGVLALFLSIVILGLMPFLHTSKHRSMMLRPLSQALFWTLTMDLLTLTWIGSQPVEYPYTIIGQMASILYFSIILAFLPIAGX IENY

FASTA Format

Sequence uploaded on course website

What does this sequence represent?

Clipping a sequence

Clipping Sequence (5’End)

Clipping Sequence (3’End)

Clipped Sequence IV

NCBI Website is Rapidly Changing

The remaining slides will help get you started for assignment 1. Keep in mind that there have been changes to the website.

Mock Sequence Submissionhttp:/www.ncbi.nlm.nih.gov/

X DO NOT HIT “DONE”

Just print the page and hand it in.

This will be page 2 ofyour assignment

http://www.ncbi.nlm.nih.gov/projects/genome/guide/bee/

The remainderof the slides will help with pages 3 & 4 of Assignment 1

http:/www.ncbi.nlm.nih.gov/

?Copy/paste your CLIPPED sequence(http://instruct.uwo.ca/biology/396a)under “Results” section on website

Group 2-7 Mar 28, 05

Additional Help and Hint

Go to “Model Maker”

The End