Chapter !3 - Mapping, Sequencing, Annotation and databases Code : KSI0012

<Index of Chapter 3>

Classical genetics as background

   - What is a gene?

   - DNA was discovered in 1869. Avery, Mcleod, and McCarty showed in 1944 that DNA was the active substance in bacterial transformation. Hershey and Chase showed that durig bacteriophafe infection, DNA but not protein entered the host cell.

 

Maps and tour guides 

   DIfferent types of map describe different types of observation

1. Linkage maps of genes.

2. Banding patterns of chromosomes.

3. Restriction maps- DNA cleavage fragment patterns

4. DNA sequences.

 

   - Genetic maps

Gene maps were classically determined from patterns of inheritance of phenotypic traits. 

 

   - Linkage

Absence or reduction of independent assortment of parental genes, which are usually transmitted together because they lie on the same chromosome

 

   - Linkage disequilibrium

The deviation of the genotype distrubution in the population from the ulimate 1:2:1 ratio is called linkage disequilibrium

 

   - Chromosome banding pattern maps

  Deletions in the region 15q11-13 are associated with prader-willi and angleman syndromes. THese syndromes have the interesting feature that alternative clinical consequences depend on whether the affected chromosome is parternal or maternal. This observation of genomic imprinting shows that the genetic information in a fertilized egg is not simply the bare DNA sequences contributed by the parents. 

    - Restriction maps

Cutting  DNA with a restriction enzyme produces a set of fragments. Cutting the same DNA with other resctriction enzymes with different specificities produces overlapping fragments. From the sizes of the gragments produced by individual enzymes and in combination , it is possible to construct a resctriction map.

 

Discovery of the structure of DNA

This attitude contributed to the general lack of accptance of the experiments of Avery, MacLeod, and Mc Carty as proof that DNa was the genetic material. 

-The chemical structure of one strand DNA. 

X-ray diffraction pattern of DNA fibre by R.Franklin. The X-shpaed pattern at the centre of the picture is diagnostic of a helical structure. From the distribution of intensity, it's possible to deduce the number of residues per turn and the symmetry of the structure. 

DNA sequencing 

   - The problem of sequence assembly from short fragments

It would be more difficult to reassemble this from fragments, because the repitions create ambiguites. Indeed the very large amounts of repetitive sequence in eukatyotic genomes do create problems for assembly algotithms. 

 

   - The Maxam-Gilbert chemical cleavage method.

As in sanger's method, polyacrtlyamide gele electrophoresiss separates the fragments by size. The separate cleavage reactions produce fragments that share the 5'-labelled end, but differ in the bases at which the specific 3'-cleavage occurs. THe sequence can be read from an autoradiograph.

 

   - Automated DNA sequencing

Using fluorescent dyes as reporters, rather than radioactivity was an important technical advance in sequencing. 

 

Organizing a large- scale sequencing project

   - Bring on the clones : hierachical - or ' BAC - to BAC' - genome sequencing 

1. Cut the DNA into fragments of about 150Kb.

Clone them into BACs.

2. Identify a series of clones in the library that contains overlapping fragments. 

3. Using the overlaps, order the clones according to their position along the original large target DNA molecule.

4. Subfragment each clone, sequence the fragments and assemble them 

   - Whole genome shotgun sequencing

reads / contig/ coverage / finishing 

   - High- throughput sequencing 

pyrosequencing detects incorporation of a specific nucleotide into a growing strand by detecting the pyrophosphate(PPi) released in a step of the reaction catalysed by polymerase.

 

Databanks in molecular biology

   - Nucleic acid sequence databases

   - Protein sequence databases

   - Databases of genetic diseases -OMIM and OMIA  

   - Databases of structures

   - Classifications of protein structures

   - Specialized or 'boutique' databases

   - Databases of metabolic pathways

   - Bibiographic databases 

   - Survery of moleucular biology databases and servers 

_______________________________________________________________________________KSI