Download A. Overview - eweb.furman.edu

Recombinant DNA Technology and Genomics
A.
B.
C.
D.
Overview:
Creating a DNA Library
Recover the clone of interest
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
A.
B.
C.
D.
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
Southern Blot allows characterization of
restriction map, and identity of
fragments with a desired sequence.
ALL BANDS
FRAG’s THAT BIND TO PROBE
Recombinant DNA Technology
A.
B.
C.
D.
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
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.
Recombinant DNA Technology
A.
B.
C.
D.
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
Recombinant DNA Technology
A.
B.
C.
D.
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
“bottom” of gel
Different reaction tubes
G
A
T
C
“bottom”
of gel
One reaction tube
G, A, T, C
A laser ‘reads’ the bands in the gel, recording the wavelengths of the
reflected light - which indicates the last base added in the fragment
Genomics
A. Overview:
PHENOTYPE
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
GENE
Genomics
A. Overview:
PHENOTYPE
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
GENE
Genomics
A. Overview:
PHENOTYPE
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
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
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.
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.
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
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.
Genomics
A. 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 cacccctacc
61 acagcacctc gggcctaggc tgggcggggg gctggggagg cagagctgcg aagaggggag
121 atgtggggtg gactcccttc cctcctcctc cccctctcca ttccaactcc caaattgggg
181 gccgggccag gcagctctga ttggctgggg cacgggcggc cggctccccc tctccgaggg
241 gcagggttcc tccctgctct ccatcaggac agtataaaag gggcccgggc cagtcgtcgg
301 agcagacggg agtttctcct cggggtcgga gcaggaggca cgcggagtgt gaggccacgc
361 atgagcggac gctaaccccc tccccagcca caaagagtct acatgtctag ggtctagaca
421 tgttcagctt tgtggacctc cggctcctgc tcctcttagc ggccaccgcc ctcctgacgc
481 acggccaaga ggaaggccaa gtcgagggcc aagacgaaga cagtaagtcc caaacttttg
541 ggagtgcaag gatactctat atcgcgcctt gcgcttggtc ccgggggccg cggcttaaaa
601 cgagacgtgg atgatccgga gactcgggaa tggaagggag atgatgaggg ctcttcctcg
661 gcgccctgag acaggaggga gctcaccctg gggcgaggtt ggggttgaac gcgccccggg
721 agcgggaggt gagggtggag cgccccgtga gttggtgcaa gagagaatcc cgagagcgca
781 accggggaag tggggatcag ggtgcagagt gaggaaagta cgtcgaagat gggatggggg
841 cgccgagcgg ggcatttgaa gcccaagatg tagaagcaat caggaaggcc gtgggatgat
901 tcataaggaa agattgccct ctctgcgggc tagagtgttg ctgggccgtg ggggtgctgg
961 gcagccgcgg gaagggggtg cggagcgtgg gcgggtggag gatgagaaac tttggcgcgg
1021 actcggcggg gcggggtcct tgcgccccct gctgaccgat gctgagcact gcgtctcccg
Genomics
A. Overview:
B. Sequencing:
C. 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’]
Genomics
A. Overview:
B. Sequencing:
C. 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):