Historical overview and mapping Flashcards
- Human Genome Project - Genetic and physical mapping
Define genetics
The study of single genes and their effects
Define genomics
The study of functions and interactions of genes in the entire genome
Define genomic medicine
The application of genomic data gained through the HGP to clinical practice, for example genetic testing.
What were the main goals of the Human Genome Project (HGP)?
- To identify all (~30,000) human genes
- To sequence the whole human genome (~3 billion bp)
- Create high resolution physical, genetic, and sequence maps
- Develop technology for sequencing DNA
- Store this information in databases
- Improve tools for data analysis
- Sequence the genome of model organisms
- Consider ethical, legal and social implications
What were the findings of the HGP?
- The human genome contains 3 billion nucleotides.
- The average gene is 3,000 base pairs long, but varies greatly with the largest being Dystrophin at 2.4 million bases.
- The total number of genes is estimated at ~30,000, down from around 100,000.
- Almost all (99.9%) of nucleotide bases are exactly the same in all people.
- The functions are unknown for over 50% of discovered genes.
What were the anticipated benefits of the HGP?
The anticipated benefits for the completion of the HGP included the improved diagnosis of disease, earlier detection of genetic predispositions to disease, better understanding of disease mechanisms, rational drug design, gene therapy and control systems for drugs, and personalised/custom drug development.
What new projects have arised after the completion of the HGP?
- HapMap
- ENCODE
- CNV projects
What were the key discoveries that set out a scientific basis for genomic medicine?
- Rediscovery of Mendel’s laws of hereditary (early 1900s).
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Cellular basis of hereditary- disovery of chromosomes and their properties (early 1900s):
- the chromosome theory of inheritance was devised by Bovery, Sutton and Morgan.
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DNA as a hereditary material (mid 1900s):
- Avery, McLeod and McCarty demonstrated that DNA is the genetic material (1944), and;
- a model of DNA was created by Watson and Crick (1953). Both of these lead to the formation of the genetic code and the central dogma of biology.
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Technical advances in DNA manipulation (later 1900s):
- Restriction enzymes act as molecular scissors for DNA.
- First recombinant DNA molecule was created by Paul Berg (1972) using SV40 virus and a lambda bacteriophage.
- DNA sequencing developed by Maxam and Gilbert (US) and Fred Sanger (UK).
- PCR acts as molecular photocopying, and was developed by Mullis (1983).
What techniques were employed during the HGP?
- Extraction of DNA: collection of cells, lysis of cells and purification using detergents, enzymes, and surfactants, salt treatment to aggregate lipids/protein/RNA, centrifugation, DNA purification using detergents/enzymes/salts.
- Fragmentation: using restriction enzymes, which recognise 6 nt pallindromic sequences and create sticky ends (naturally found in bacteria to protect against viruses).
- Recombinant DNA technology: the fragmented DNA was used to make a genomic DNA library containing the whole 3 billion base pairs of DNA. YACs are preferable as they can take the largest inserts (<1 Mb).
- Mapping: the addition of markers on the DNA allowed for mapping of sequences to chromosome loci using FISH.
- PCR was used to amplify the DNA.
- Sequencing: used after the mapping was dense enough to relate different stretches of DNA to single nucleotide accuracy. This was done using Sanger sequencing (See image. This is now outdated, we now use Next Generation Sequencing).
Outline DNA variation.
Genetic variation is a result of subtle differences in our DNA, i.e. when there are differences in nucleotide sequence at the same locus. DNA sequence variation exists between members of the same species, for example different alleles of the same gene. Variations are termed polymorphisms and when a particular polymorphic locus is useful for DNA mapping it’s called a DNA marker. These include SNPs, microsatellites (<100 bp) and minisatellites (0.1 - 20 kb).
What are the different types of DNA markers?
- SNPs
- Microsatellites (<100 bp)
- Minisatellites (0.1 - 20 kb)
Outline the differences between microsatellites and minisatellites.
Both are sequences that resemble satellites, but are much shorter in length (5-50 repeats), and are common in mammalian genomes with their length varying considerably in human DNA. They are also called variable number tandem repeats (VNTRs).
- Microsatellite is used to describe a repeating unit with a length of <10 bp, and tend to be <100 bp in total.
- Microsatellites are tandem repeats with individual units between 10-100 bp, spanning from 0.1-20 kb in total.
Outline what a genetic map is
A genetic map is a map that relies on tracing inheritance of markers through generations. Polymorphic loci can be positioned relative to each other based upon the frequency with which they recombine in meiosis. More comprehensive genetic maps can be made using RFLPs.
- Limitations: the average spacing between markers too large
Define restriction fragment length polymorphism (RFLP)
Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination.
The RFLP probes are frequently used in genome mapping.
Define genome mapping
assigning/locating of a specific gene to particular region of a chromosome and determining the location of and relative distances between genes on the chromosome.
What are the two forms of human genome maps? Outline each.
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Linkage maps (genetic maps): show the arrangement of genes and genetic markers along the chromosomes as calculated by the frequency with which they are inherited together (recombination frequencies). Linked markers tend to be co-inherited, and the closer they are the higher the rate of co-inheritance.
- In more complex diseases (common diseases) mapping is based on SNPs. Haplotype blocks are analysed (HapMap charted the location of these blocks).
- Physical maps: represent chromosomes and provide physical distances between chromosomal landmarks ideally measured in nucleotide bases. They are constructed by plotting the location of physical mapping markers, such as sequence tagged sites. Now the genome has been fully sequenced and labelled, detailed physical maps are available in database such as Ensembl.
Define contig
Contiguous sequence of DNA created by assembling overlapping sequenced fragments of a chromosome. A group of clones representing overlapping regions of the genome.
