Recombinant DNA Technology and Genomics Overview: Creating a DNA Library

1. Recombinant DNA Technology and Genomics • Overview: • Creating a DNA Library • Recover the clone of interest • Analyzing/characterizing the DNA • - create a restriction map

2. Restriction maps can be used to identify DNA sequences, because different sequences, like different alleles, should have these random sequences in different positions. As in p53 lab, alleles were identified by differences in their cleavage pattern!!! Also used in DNA fingerprinting

3. Recombinant DNA Technology • Overview: • Creating a DNA Library • Recover the clone of interest • Analyzing/characterizing the DNA • - create a restriction map • - combine restriction mapping and clone isolation – Southern Blot

4. Southern Blot allows characterization of restriction map, and identity of fragments with a desired sequence.

5. ALL BANDS FRAG’s THAT BIND TO PROBE

6. Recombinant DNA Technology • Overview: • Creating a DNA Library • Recover the clone of interest • Analyzing/characterizing the DNA • - create a restriction map • - combine restriction mapping and clone isolation – Southern Blot • - assessing gene activation – Northern Blot

7. In a ‘Northern Blot’, m-RNA is run, and a DNA probe is used. Used to describe patterns of gene activity (m-RNA production) in cells.

8. Recombinant DNA Technology • Overview: • Creating a DNA Library • Recover the clone of interest • Analyzing/characterizing the DNA • - create a restriction map • - combine restriction mapping and clone isolation – Southern Blot • - assessing gene activation – Northern Blot • - DNA sequencing

9. Recombinant DNA Technology • Overview: • Creating a DNA Library • Recover the clone of interest • Analyzing/characterizing the DNA • - create a restriction map • - combine restriction mapping and clone isolation – Southern Blot • - assessing gene activation – Northern Blot • - DNA sequencing Sanger Method: Logic – DNA to be sequenced is denatured, and used as a template for the formation of new DNA. A Primer is used to begin synthesis. Synthesis is terminated by the incorporation of di-deoxy bases that lack a –OH on 3’ end. So, when they are incorporated, synthesis stops. Electrophoresis separates the fragments

11. “bottom” of gel Different reaction tubes G A T C

12. One reaction tube “bottom” of gel G, A, T, C

13. A laser ‘reads’ the bands in the gel, recording the wavelengths of the reflected light - which indicates the last base added in the fragment

14. Genomics • Overview: • The history of understanding genome structure and function has been largely a “top-down” process, by correlating changes in the phenotype with the inheritance of a particular gene. • - the down sides are: • 1) can’t ‘cross’ most species • 2) mutants can be very rare • 3) mutations may be silent • 4) building recombination maps • is laborious PHENOTYPE GENE

15. Genomics A. Overview: The ability to sequence DNA allows for a bottom up approach – sequencing genes and then trying and figure out what they do at a phenotypic level. - the benefits are: 1) All species can be studied 2) The techniques are not difficult PHENOTYPE GENE

16. Genomics • Overview: • The ability to sequence DNA allows for a bottom up approach – sequencing genes and then trying and figure out what they do at a phenotypic level. • - the benefits are: • 1) All species can be studied • 2) The techniques are not difficult • - there are still significant hurdles: • 1) most DNA is non-coding; • finding genes is hard • 2) linking a coding sequence to a • function is difficult PHENOTYPE Knowing the sequence of A, T, C, G in a genome is just the beginning, and does not answer the fundamental question of how a genome encodes a phenotype. GENE

17. Genomics A. Overview: B. Sequencing: - Basically, you sequence the longest fragments of DNA that you can, by the methods we have described already. - Then, you enter the sequence in a computer, and you group together “contiguous sequences” (contigs) based on regions of overlap. Eventually, you cover the entire map.

18. Genomics A. Overview: B. Sequencing: - Clone-by-Clone Method: Once you have a restriction map of a chromosome, you can partially digest with different combinations of enzymes to make overlapping clones that are inserted into BAC’s or YAC’s or cosmids or plasmids and then replicated in cell clones and sequenced by the Sanger Method.

19. Genomics A. Overview: B. Sequencing: - Clone-by-Clone Method: Once you have a restriction map of a chromosome, you can partially digest with different combinations of enzymes to make overlapping clones that are inserted into BAC’s or YAC’s or cosmids or plasmids and then replicated in cell clones and sequenced by the Sanger Method. Reconstruction occurs by matching overlapping ‘contiguous sequences’. LABOR INTENSIVE – can only sequence ~300 bases/gel

20. Genomics A. Overview: B. Sequencing: - Whole Genome Shotgun: Initially the same approach; partial digests create overlapping fragments. High-throughput Automated Sequencers use dd-NTP’s and capillary tubes several feet long as the single lane ‘gels’, able to sequence 900 base fragments. The sequencer may run 96 capillary tubes at a time, sequencing nearly 2 million bases/day. Contig sequences analyzed by computer, and a sequence map is created.

21. Genomics • A. Overview: • B. Sequencing: • Finding Genes – structural genomics and ‘annotation’: • - once you have the sequence data, you really have just started. • - The goals are then: • - identify where genes are (Open Reading Frames) • - find promoters and regulatory elements to confirm this is a gene (and not a pseudogene). • - in eukaryotes, find splice sites, introns and exons • - identify structural sequences like telomeres and centromeres • - convert the DNA sequence into the predicted AA sequence of the protein • - predict protein structure and function by identifying ‘domains’ and ‘motifs’ • - These goals are attained by computer analyses of gene/AA sequence data, and comparison with known described genes. This is: BIOINFORMATICS

22. Genomics • A. Overview: • B. Sequencing: • Finding Genes – structural genomics and ‘annotation’: • 1. NCBI – BLAST search compares sequence to other sequences in the database 1 ggggcacccc tacccactgg ttagcccacg ccatcctgag gacccagctg cacccctacc61 acagcacctc gggcctaggc tgggcggggg gctggggagg cagagctgcg aagaggggag121 atgtggggtg gactcccttc cctcctcctc cccctctcca ttccaactcc caaattgggg181 gccgggccag gcagctctga ttggctgggg cacgggcggc cggctccccc tctccgaggg241 gcagggttcc tccctgctct ccatcaggac agtataaaag gggcccgggc cagtcgtcgg301 agcagacggg agtttctcct cggggtcgga gcaggaggca cgcggagtgt gaggccacgc361 atgagcggac gctaaccccc tccccagcca caaagagtct acatgtctag ggtctagaca421 tgttcagctt tgtggacctc cggctcctgc tcctcttagc ggccaccgcc ctcctgacgc481 acggccaaga ggaaggccaa gtcgagggcc aagacgaaga cagtaagtcc caaacttttg541 ggagtgcaag gatactctat atcgcgcctt gcgcttggtc ccgggggccg cggcttaaaa601 cgagacgtgg atgatccgga gactcgggaa tggaagggag atgatgaggg ctcttcctcg661 gcgccctgag acaggaggga gctcaccctg gggcgaggtt ggggttgaac gcgccccggg721 agcgggaggt gagggtggag cgccccgtga gttggtgcaa gagagaatcc cgagagcgca781 accggggaag tggggatcag ggtgcagagt gaggaaagta cgtcgaagat gggatggggg841 cgccgagcgg ggcatttgaa gcccaagatg tagaagcaat caggaaggcc gtgggatgat901 tcataaggaa agattgccct ctctgcgggc tagagtgttg ctgggccgtg ggggtgctgg961 gcagccgcgg gaagggggtg cggagcgtgg gcgggtggag gatgagaaac tttggcgcgg1021 actcggcggg gcggggtcct tgcgccccct gctgaccgat gctgagcact gcgtctcccg

23. Genomics • A. Overview: • B. Sequencing: • Finding Genes – structural genomics and ‘annotation’: • 1. NCBI – BLAST search compares sequence to other sequences in the database • 2. Open Reading Frames: base sequences which would code for long stretches of AA’s before a stop codon would be reached. Typically, these are found by looking for [5’ – ATG…-3’] sequences that follow a promoter (TATA, CAAT, GGGCGG). The complement would be [3’ – TAC..-5’], which would encode a start codon in RNA [5’- AUG…3’]

24. Genomics • A. Overview: • B. Sequencing: • Finding Genes – structural genomics and ‘annotation’: • 1. NCBI – BLAST search compares sequence to other sequences in the database • 2. Open Reading Frames: base sequences which would code for long stretches of AA’s before a stop codon would be reached. Typically, these are found by looking for [5’ – ATG…-3’] sequences that follow a promoter (TATA, CAAT, GGGCGG). The complement would be [3’ – TAC..-5’], which would encode a start codon in RNA [5’- AUG…3’] • 3. Regulatory regions and splicing sites (GT-AG):