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which one happens is random, based on ratio of dNTP to ddNT

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which one happens is random, based on ratio of dNTP to ddNTP in the tube. ... Zoo blot: a Southern blot containing genomic DNA from many species. ... – PowerPoint PPT presentation

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Title: which one happens is random, based on ratio of dNTP to ddNT


1
DNA Sequencing and Gene Analysis
2
Determining DNA Sequence
  • Originally 2 methods were invented around 1976,
    but only one is widely used invented by Fred
    Sanger.
  • Uses DNA polymerase to synthesize a second DNA
    strand that is labeled. Recall that DNA
    polymerase always adds new bases to a primer.
  • Also uses chain terminator nucleotides dideoxy
    nucleotides (ddNTPs), which lack the -OH group on
    the 3' carbon of the deoxyribose. When DNA
    polymerase inserts one of these ddNTPs into the
    growing DNA chain, the chain terminates, as
    nothing can be added to its 3' end.

3
Sequencing Reaction
  • The template DNA is usually single stranded DNA,
    which can be produced from plasmid cloning
    vectors that contain the origin of replication
    from a single stranded bacteriophage such as M13
    or fd. Infecting bacteria containing this vector
    with a helper phage causes single stranded
    phage to be produced. The phage DNA contains the
    cloned insert
  • The primer is complementary to the region in the
    vector adjacent to the multiple cloning site.
  • Sequencing is done by having 4 separate
    reactions, one for each DNA base.
  • All 4 reactions contain the 4 normal dNTPs, but
    each reaction also contains one of the ddNTPs.
  • In each reaction, DNA polymerase starts creating
    the second strand beginning at the primer.
  • When DNA polymerase reaches a base for which some
    ddNTP is present, the chain will either
  • terminate if a ddNTP is added, or
  • continue if the corresponding dNTP is added.
  • which one happens is random, based on ratio of
    dNTP to ddNTP in the tube.
  • However, all the second strands in, say, the A
    tube will end at some A base you get a
    collection of DNAs that end at each of the A's in
    the region being sequenced.

4
Electrophoresis
  • The newly synthesized DNA from the 4 reactions is
    then run (in separate lanes) on an
    electrophoresis gel.
  • The DNA bands fall into a ladder-like sequence,
    spaced one base apart. The actual sequence can
    be read from the bottom of the gel up.
  • Automated sequencers use 4 different fluorescent
    dyes as tags and run all 4 reactions in the same
    lane of the gel.
  • Radioactive nucleotides (32P) are used for
    non-automated sequencing.
  • Sequencing reactions usually produce about 500 bp
    of good sequence.

5
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6
Single Nucleotide Polymorphisms
  • Looking at many individuals, you can see that
    most bases in their DNA are the same in everyone.
    However, some bases are different in different
    individuals. These changes are single nucleotide
    polymorphisms (SNPs).
  • SNPs are found everywhere in the genome, and they
    are inherited in a regular Mendelian fashion.
    These characteristics makes them good markers for
    finding disease genes and determining their
    inheritance.
  • Lots of ways to detect SNPs, many of which are
    easy to automate.
  • Primer extension make a primer 1 base short of
    the SNP site, and then extend the primer using
    DNA polymerase with nucleotides having different
    fluorescent tags.

7
Gene Detection
  • It is surprisingly hard to be sure that a given
    genomic sequence is a gene that it is ever
    expressed as RNA.
  • Protein-coding regions are open reading frames
    (ORFs) they dont contain stop codons.
  • But, human genes often contain long introns and
    very short exons, and some parts of genes are
    introns in one cell type but exons in other cell
    types. So, finding all the pieces of a gene can
    be a challenge.
  • Three questions
  • is a given DNA sequence ever expressed?
  • is the sequence expressed in a given cell type or
    set of conditions?
  • what is the intron/exon structure of the sequence?

8
Evolutionarily Conserved Sequences
  • When looking across different species, most DNA
    sequences are not conserved.
  • However, the exons of genes are often highly
    conserved, because their function is necessary
    for life.
  • Zoo blot a Southern blot containing genomic DNA
    from many species. Probe it with the sequence in
    question exons will hybridize with other
    species DNA, while introns and non-gene DNA
    wont.
  • Computer-based homology search BLAST search. Do
    similar sequences appear in the nucleotide
    databases? Especially chimpanzee and mouse,
    which have complete genome sequences available.

9
Detecting Gene Expression
  • Northern blots RNA extracted from various
    tissues or experimental conditions, run on an
    electrophoresis gel, then probed with a specific
    DNA sequence.

10
Detecting Gene Expression
  • Real time PCR
  • first convert all mRNA in a sample to cDNA using
    reverse transcriptase,
  • then amplify the region of interest using
    specific primers.
  • Use a fluorescent probe to detect and quantitate
    the specific product as it is being made by the
    PCR reaction.
  • the two components of the fluorescent tag
    interact to quench each other. When one part is
    removed by the Taq polymerase, the quenching
    stops and fluorescence can be detected.

11
Expressed Sequence Tags
  • ESTs are cDNA clones that have has a single round
    of sequencing done from one end.
  • First extract mRNA from a given tissue. Then
    convert it to cDNA and clone.
  • Sequence thousands of EST clones and save the
    results in a database.
  • A search can then show whether your sequence was
    expressed in that tissue.
  • quantitation issues some mRNAs are present in
    much higher concentration than others. Many EST
    libraries are normalized by removing duplicate
    sequences.
  • Also can get data on transcription start sites
    and exon/intron boundaries by comparing to
    genomic DNA
  • but sometimes need to obtain the clone and
    sequence the rest of it yourself.

12
Etc.
  • New techniques in DNA/RNA technology are being
    developed constantly. The main goal is to
    increase reliability and decrease cost.
    Primarily the aim is to automate as much as
    possible.
  • Just a few techniques we are not going to
    discuss RACE, SAGE, differential display, S1
    nuclease protection

13
Protein Methods
  • It is important to be sure that the protein
    product of a gene is made, and to know where in
    the tissue or cell it is made, and how much is
    made.
  • Most protein detection is based on either making
    antibodies to the protein of interest, or by
    making a fusion protein your protein fused to a
    fluorescent protein.
  • GFP green fluorescent protein. Isolated from
    jellyfish. Several variants give different
    colors. It still works when it is fused to other
    proteins.
  • Often done in conjunction with confocal
    microscopy examining the same image with visible
    light and fluorescence.

14
Antibodies
  • If you inject rabbits (usually) with your
    protein, the rabbit will develop an immune
    response against it. The antibodies can be
    isolated from the blood.
  • Antibodies bind very specifically to the antigen.
    The antibodies can be detected by a labeled
    second antibody that binds to the first antibody
    fluorescein-labeled goat anti-rabbit antibody for
    instance.
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