Recombinant DNA Technology and Genomics Overview: Creating a DNA Library

Recombinant DNA Technology and Genomics

A.Overview:B.Creating a DNA LibraryC.Recover the clone of interestD.Analyzing/characterizing the DNA

- create a restriction map

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

Recombinant DNA Technology


- create a restriction map - combine restriction mapping and clone isolation – Southern Blot

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

ALL BANDS FRAG’s THAT BIND TO PROBE



- create a restriction map - combine restriction mapping and clone isolation – Southern Blot - assessing gene activation – Northern Blot

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.



- create a restriction map - combine restriction mapping and clone isolation – Southern Blot - assessing gene activation – Northern Blot - DNA sequencing



- 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

G A T C

Different reaction tubes “bot

tom

” of

gel

G, A, T, C

One reaction tube

“bot

tom

” of

gel

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

GenomicsA.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 species2) mutants can be very rare3) mutations may be silent4) building recombination maps is laborious

PHENOTYPE

GENE

GenomicsA. 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 studied2) The techniques are not difficult

PHENOTYPE

GENE

GenomicsA.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 studied2) The techniques are not difficult

- there are still significant hurdles:1) most DNA is non-coding; finding genes is hard2) linking a coding sequence to a function is difficult

PHENOTYPE

GENE

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.

GenomicsA. 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.


- 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.


- 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


- 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.

GenomicsA. Overview:B. Sequencing:C.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

Genomics

A. Overview:B. Sequencing:C.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

Genomics



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’]

Genomics



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):

Documents

Recombinant DNA Technology and Genomics Overview: Creating a DNA Library