Molecular%20Basis%20for

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Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
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Reading the DNA sequence from an automatic
sequencer
Refer to Figure 10-18, Griffiths et al., 2015.
Oligonucleotide primers can be tagged with
fluorescent dyes instead of radioactive labels.
A different colored dye can be used for each of
the four reactions.
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In Search of Potential Genes
Open reading frames (ORFs) are long stretches of
DNA that start with ATG and end with a stop
codon. A double-stranded DNA molecule has 6
possible reading frames, 3 for each strand.
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Molecular Genetic Diagnostics
Restriction Site Analysis Mutant allele and
wild-type allele could differ in presence or
absence of restriction site. (e.g., sickle cell
anemia) Probe Hybridization Synthetic probe
could distinguish between mutant and wild-type
alleles by hybridization at elevated
temperatures. (e.g., a1-antitrypsin
deficiency) PCR Tests Primers can be designed
to hybridize with only wild-type allele sequence
such that mutant allele will not be amplified.
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Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
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Detection and Isolation of Target Molecules
For Nucleic Acids 1. cDNA of a specifc gene 2.
Homologous DNA of gene from related
organism 3. Synthetic DNA based on amino acid
sequence of protein For Proteins 1. Antibodies
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(No Transcript)
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Separation of Molecules by Gel Electrophoresis
Subject molecules to electrical field in a
matrix. Separation of molecules is based
on 1. net charge 2. size 3. shape
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Blotting Techniques
Southern Analysis DNA fragments are
electrophoresed and probed with DNA or cDNA for
specific sequence. Northern Analysis RNA
molecules are electrophoresed and probed with
cDNA for specific sequence. Western
Analysis Proteins are electrophoresed and probed
with antibody for specific protein.
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Southern Analysis
1. Fragmented DNA molecules are separated by gel
electrophoresis. 2. After electrophoresis, gel
is placed in container of buffer and
nitrocellulose filter is placed on top of the gel
to transfer DNA by capillary action to the
filter. 3. Filter is incubated with labeled
single-stranded probe (often radioactive probe is
used). 4. After unbound probe is removed, the
filter is placed on an X-ray film for
autoradiography. 5. Probe hybridizes only with
complementary fragments.
Southern blotting
Gel stained with ethidium bromide.
Autoradiogram
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Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
12
25,000 genes in humans are potential candidates
for gene therapy.
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Molecular Genetic Diagnostics Sickle Cell Anemia
Electron Micrograph HbA codes for normal
b-globin and produces normal hemoglobin. HbS
produces sickled red blood cells. Homozygotes
for HbS are anemic. HbS produces b-globin that
differs from normal protein by one amino acid.
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Molecular Genetic Diagnostics Sickle Cell Anemia
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Molecular Genetic Diagnostics Sickle Cell Anemia
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Methods of introducing a transgene
Solution of DNA to be studied is injected into
host cell.
Recombinant DNA can be delivered into host cell
using a viral vector.
Refer to Figure 10-23, Griffiths et al., 2015.
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Methods of introducing a transgene
Bombardment with DNA-coated tungsten particles
introduces DNA into host cell.
Under favorable conditions, DNA can be taken up
by host cell by transformation.