Lecture 11: Recombinant DNA Technology in Genetics

Lectures 12-15: Molecular Genetics of Human Disease: Hemoglobinopathies

Lectures 21-22: Cancer Genetics

Lectures 23-25: Gene Mapping

Lecture 30: Gene Therapy


Lecture 11: Recombinant DNA Technology in Genetics


1) Know the general structure of a gene and the steps in gene expression.

2) Be familiar with the basic methods of molecular biology used in genetics:

  • Southern and northern blotting

    • PCR
    • DNA sequencing
    • basic steps involved in constructing a cDNA library
    • nucleic acid hybridization
    • screening a cDNA library

3) Understand the meaning and significance of DNA sequence and amino acid polymorphisms.

4) Understand the molecular basis for the various types of commonly used DNA sequence polymorphisms.

    • RFLPs
    • VNTRs
    • SSRs
    • SNPs

5) Understand how DNA sequence analysis is performed and be familiar with methods of screening for differences.

  • ASO
  • DGGE
  • SSCP
  • Chemical mismatched cleavage

6) Have a general understanding of methods for gene transfer into tissue culture cells and the power of transgenic technologies.


Lectures 12-15: Molecular Genetics of Human Disease: Hemoglobinopathies


You should be familiar with the basic structure and function of the hemoglobin genes and the diseases resulting from mutations in these genes. The basic principles covered here serve as a paradigm for understanding all human genetic diseases. Though you will not be expected to recall detailed information about the clinical presentation and treatment of sickle cell anemia and the thalassemias, you should be familiar with the general principles and in particular be able to apply knowledge of the molecular defect to understanding the clinical disease.

1) Be familiar with the developmental pattern of expression of the human globins, particularly the fetal to adult globin switch.

2) Understand the general structure of the prototypical b-globin gene and how it is expressed.

3) Understand the concept of globin chain imbalance that underlies the a and b-thalassemias.

4) Be able to distinguish a- and b-thalassemia, both in terms of the general clinical picture and molecular basis.

5) Know the two major types of deletions that can cause a-thalassemia and how they interact to cause the gradation of a-thalassemia phenotypes.

6) Understand how the range of severity from mutations in the b-globin locus can result in varying b-thalassemia phenotypes. Understand the multiple different molecular mechanisms that can cause the thalassemias (and other genetic diseases) including:

    • Unequal crossing over
    • Gene deletion
    • Mutations in the promoter and other transcriptional regulatory sequences
    • Mutations that effect splicing; (Be able to describe several mechanisms by which mutations can interfere with normal splicing and how this can cause partial or complete loss of gene function.)
    • Nonsense and frameshift mutations
    • PolyA site mutations
    • Mutations in the initiator codon and cap site

7) Be particularly familiar with the molecular basis for sickle cell anemia.

    • The genetic origins of the sickle mutation
    • Interaction with other globin gene abnormalities, including hemoglobin and thalassemia
    • How the mutant protein and DNA change can be detected and how this information is used for pre- and postnatal diagnosis

8) Be familiar with the syndrome of HPFH and the general classes of mutations that can produce this disease.


Lectures 21-22: Cancer Genetics


1) Understand that cancer is a genetic disease.

2) Understand the significance of the clonal nature of cancer and be familiar with several types of evidence for clonality.

3) Be familiar with oncogenes.

    • Understand how these genes act in a dominant transforming way.
    • Know how this information was used to initially clone and identify these genes.
    • Understand the relationship between transforming retroviruses and oncogenes.
    • Understand the relationship between the normal cellular proto-oncogene and the mutant forms ("oncogenes") that contribute to cancer.

4) Know the mechanisms that can result in oncogene activation.

    • Point mutation
    • Amplification
    • Chromosome translocation

5) Be familiar with the concept of tumor suppressor genes.

    • Be able to describe Knudson's hypothesis, using sporadic and familial retinoblastoma as an example.
    • Understand the various mechanisms for the "second hit" in a tumor suppressor gene already carrying a germline mutation in a familial cancer syndrome. Be able to describe how these different mechanisms would appear when studied with genetic markers in the critical chromosomal region.

  • Understand in general terms how this information has been used to clone familial cancer genes.

6) Understand current models for the multi-step process of cancer development as exemplified by colon cancer. Know the two general classes of familial colon cancer syndromes, familial adenomous polyposis and hereditary nonpolyposis colon cancer, and the types of gene mutations that cause each one.

7) Be familiar with other common familial cancer syndromes, particularly familial breast cancer.

8) Be able to combine this information with an understanding of gene mutation mechanisms and molecular genetic techniques discussed earlier in the class to describe how cancer predisposition gene mutations can be detected.

a) Understand the major technical, ethical, and social issues involved in DNA testing for cancer predisposition syndromes.


Lectures 23-25: Gene Mapping


1) Be familiar with the concept of recombination and how it allows genes to be mapped.

2) Understand the concept of linkage. Understand in general terms the significance of a LOD score, though it is NOT necessary to know how to directly calculate one.

3) Understand how a marker locus linked to a disease gene can be used for genetic diagnosis, as well as for identifying the gene by positional cloning.

4) Know the difference between a polymorphic linked marker and the disease causing mutation itself.

5) Be able to define

    • Phase

  • Centimorgan

    • Physical map
    • Genetic map

6) Be able to look at genotype data for a pedigree and assign phase to define haplotypes (and know what all that means). Understand the meaning of haplotype and linkage disequilibrium.

7) Be familiar with several methods, in addition to linkage, that are used to map genes, including in situ hybridization and the analysis of somatic cell hybrids.

8) Understand the difference between functional and positional cloning. Know in general terms the steps involved in positional cloning. Be able to discuss the types of evidence necessary to prove the identity of a disease-causing gene.

9) Understand the overall goals and recent progress of the Human Genome Project. Be familiar with the rapidly expanding computer resources that are available for clinical and basic genetics.

10) Have a general understanding of the importance and technical difficulties inherent in defining the genetics of complex human diseases.



Lecture 30: Gene Therapy


1) Understand the difference and distinction between germline gene therapy and somatic gene therapy.

2) Be familiar with the general classes of vectors currently used in gene therapy and be able to describe their relative advantages and disadvantages including:

    • Retrovirus
    • Adenovirus
    • Adeno-associated virus
    • Non-viral vectors

3) Understand the difference between in vivo and ex vivo gene therapy.

4) Have a general familiarity with the current status of human gene therapy experimentation.

5) Understand the potential of pharmaceuticals produced by recombinant DNA technology.