Bio3400 Chapter 22 Applications and Ethics of Biotechnology
  1. Genetic disorders can be diagnosed prenatally by                followed by restriction fragment length               (RFLP) diagnosis.

      Amniocentesis is used to extract fetal cells for genetic analysis. The position of the fetus is first determined by ultrasound; a needle is then inserted through the abdominal and uterine walls to recover amniotic fluid and fetal cells.

      Homologous chromosomes can be distinguished by restriction fragments if their nucleotide sequences differ in the restriction sites. On chromosome A, three cutting sites generate fragments of 7 kb and 3 kb. On chromosome B, two cutting sites generate a single 10 kb fragment. The possible genotypes, AA, AB, and BB, produce restriction fragment-length polymorphisms (RFLPs), which can be identified on a Southern blot.

      Restriction fragment length polymorphism to detect sickle-cell anemia. The normal allele has a GAG sequence recognized by the MstII restriction enzyme, produces 2 fragments on a Southern blot, while the mutant b^s allele contains a GTG sequence resulting in a single large fragment. In this pedigree, II-1 is homozygous normal, II-2 is homozygous recessive, and II-3 is a heterozygous carrier.
  2. Allele-specific                   (ASOs) can be used to identify alleles that differ by a single nucleotide, without using radioactive isotopes..

      Synthetic allele-specific oligonucleotides (ASOs) can identify alleles that differ by a single nucleotide, such as the sickle-cell trait. The b-globin gene is amplified by PCR and denatured, then hybridized to radioactive ASO probes on filters for autoradiography. This ASO is made from the normal (A) b-globin allele (containing a GAG codon); AA-homozygous individuals show heavy hybridization; AS-heterozygous individuals show weaker hybridization; and SS sickle-cell individuals show no hybridization. Continue

      This ASO probe is made from the sickle-cell (S) b-globin allele (note the mutant GTG codon); no hybridization is shown by the AA genotype, weak hybridization by the heterozygote (AS), and strong hybridization by the homozygous sickle-cell(SS).

      Allele-specific oligonucleotides (ASOs) synthesized from normal and mutant alleles and cloned by PCR can screen for the D508 deletion in cystic fibrosis (CF). DNA from the parents (I-1 and I-2) hybridizes to both ASOs, indicating that they are heterozygous. DNA from II-1 hybridizes only to the D508 ASO, indicating that she is homozygous for the mutation and has CF.
    • ASOs have been used to produce DNA              in which expression of thousands of genes can be examined on a single chip.

        To make a DNA microarray (DNA chip), allele-specific oligonucleotides (ASOs) are attached to fields on a glass substrate. Using semiconductor technology, hundreds or thousands of genes can be tested on a single chip.

        DNA microarray assay.
      • DNA for 5 genes are amplified by PCR, denatured, and tagged with fluorescent dyes.
      • The DNA is pumped into a microarray (DNA chip) containing ASO probes for 9 alleles.
      • The hybridization is analyzed by laser and computer software
      • The color patterns show the patient's genotype.
    •              can be used to detect some forms of cancer.

        cDNA produced from normal and cancer cells can be analyzed on a DNA microarray. Green dots in this DNA microarray represent genes expressed in normal cells. Red dots represent genes expressed in cancer cells. Intermediate colors represent intermediate levels of expression.
    • Genome           using microarrays that carry all the genes in the human         makes it possible to analyze an individual's DNA for many disease alleles.

      Genome Scanning. This microarray, called a GeneChip, contains 50,000 human genes. It can be used to scan a patient's genome for disease alleles associated with heart attacks, diabetes, Alzheimer disease, etc, and to assess the patient's risks for developing genetic disorders.
  3. Genes can be mapped directly to metaphase chromosomes using fluorescent in situ                (FISH).

      Fluorescent In Situ Hybridization (FISH). Metaphase chromosomes are denatured into single-stranded DNA molecules. A probe (DNA with known sequence) labeled with fluorescent dyes is hybridized to the chromosome preparation, which is then inspected with a microscope. Continue.

      Fluorescent In Situ Hybridization (FISH) can directly map genes on a chromosome. Here 4 clusters of 2 different DNA sequences (DAZG5 and DAZG2) are mapped along a chromosome fiber stretched along a slide. The resolution can be down to 100 kb.
  4. Moderately repetitive DNA sequences in the human genome called                 (VNTRs) and                  (STRs) provide polymorphisms for use as genetic markers.

      Most eukaryotic DNA exhibit uniform density (indicating similar G-C/A-T rations), and are detected in a main band when analyzed with sedimentation equilibrium centrifugation. Often, a satellite band with a different density is also observed, containing repetitive DNA sequences.

      Insedimentation equilibrium centrifugation (or density gradient centrifugation), different molecules in the mixture will settle in bands of different buoyant densities where the centrifugal force is equal and opposite to the upward diffusion force. The gradient is eluted from the tube in fractions, which can then be measured for UV absorption at 260 nm. This technique can be used to analyze base composition of double-stranded DNA.

      centromeric DNA. Satellite DNA is highly repetitive, and exhibits fast reassociation kinetics. They are found in heterochromatic regions near the centromeres, and can be localized on an autoradiograph by in situ hybridization with a radioactive probe.
    • Variable-number         repeats (VNTRs) can be used as probes for DNA                 .

        Variable-number tandem repeats (VNTRs, or minisatellites) contain repeating clusters of 10-100 nucleotides that are variable between individuals, and may produce different restriction fragments. Even though one band (B2) is shared between 2 individuals in this RFLP analysis of many VNTR markers (this example shows just 2 markers) can serve as a DNA fingerprint to identify individuals. Relatively large sample of DNA (10,000 cells) is required for VNTR analysis, so this technoqie is more useful in paternity testing than in criminal cases.
    • Short         repeats (STRs) are used for forensic work to generate DNA profiles from trace samples.

      Small amounts of short tandem repeats (STRs, or microsatellites), consisting of 2-9 bp repeats, can be collected from crime scenes and amplified by PCR to produce DNA fingerprints. This forensic analysis shiws patterns from the victim, the specimen, and 3 suspects in a criminal case.
    • Which individual's "fingerprint" matches that from the specimen?
    • Suspect 1