Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

Dideoxy DNA Sequencing Chain-terminating (dideoxy) nucleotide Use of dideoxy nucleotide in primer extension reaction will randomly arrest DNA synthesis.

Dideoxy DNA Sequencing 4 different reactions are conducted, each with a different type of dideoxy nucleotide. Fragments are separated by electrophoresis. Banding pattern is used to infer the base sequence of the original template strand.

Sequencing Gel Using RadioactivePrimerRemember… This is base sequence of synthesized strand. Migration 3’3’ 5’5’

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. Reading the DNA sequence from an automatic sequencer

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.

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.,  1 -antitrypsin deficiency) PCR Tests: Primers can be designed to hybridize with only wild- type allele sequence such that mutant allele will not be amplified.

Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

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

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

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.

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. Gel stained with ethidium bromide. Autoradiogram Southern blotting

Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

~ 25,000 genes in humans are potential candidates for gene therapy.

Molecular Genetic Diagnostics: Sickle Cell Anemia Electron Micrograph Hb A codes for normal  -globin and produces normal hemoglobin. Hb S produces sickled red blood cells. Homozygotes for Hb S are anemic. Hb S produces  -globin that differs from normal protein by one amino acid.

Molecular Genetic Diagnostics: Sickle Cell Anemia

Solution of DNA to be studied is injected into host cell. Recombinant DNA can be delivered into host cell using a viral vector. Methods of introducting 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. Methods of introducting a transgene