Published on September 16, 2014
CONSTRUCTION OF HUMAN GENE MAP THROUGH MAP INTEGRATION- FROM GENETIC MAP TO PHYSICAL MAP TO SEQUENCE MAP Preety Sweta Hembrom M.Sc Genomic Science
MAP INTEGRATION Mapping is identifying relationships between genes on chromosomes Two broad categories of map: 1. Genetic map 2. Physical map
GENETIC MAP Describes the order of genes or other markers and the spacing between them on each chromosome. Use of genetic markers. DNA based marker can also serve as markers. Value of genetic map is that an inherited disease can be located on the map. Used to find the exact location of several important disease genes.
1. RFLPS (RESTRICTION FRAGMENT LENGTH POLYMORPHISMS): Defined by the presence or absence of a specific site- restriction site. If 2 related but different DNA molecules are cut with the same restriction enzymes, a segment of different lengths are produced. And RFLP is the difference between two DNA sequences that affect a restriction site.
2. SIMPLE SEQUENCE LENGTH POLYMORPHISMS (SSLPS) Arrays of repeat sequences that display length variations, different alleles containing different numbers of repeat units. Two types of SSLP: I. Minisatellites:- Also known as variable number of tandem repeats (VNTRs) Defined by the presence of a nucleotide sequence that is repeated several times.
II. MICROSATELLITES simple tandem repeats (STRs) Whose repeats are shorter, usually dinucleotide or tetra nucleotide units. Polymorphic because the number of repeats may vary. Scored by determining their length by PCR. Fragments are separated by electrophoresis. Primers is labeled with fluorescent dye. Human genome contains 5870 markers.
3. SNPS (SINGLE NUCLEOTIDE POLYMORPHISMS) Positions in a genome where some individuals have one nucleotide and others have a different nucleotide. Some of which also give rise to RFLPs. In the human genome there are at least 1.42 million SNPs, only 100 000 of which result in an RFLP.
LOD SCORE METHOD FOR ESTIMATING RECOMBINATION FREQUENCY Imperfect pedigrees are analyzed statistically, using a measure called the lod score (Morton, 1955). This stands for logarithm of the odds that the genes are linked and is used primarily to determine if the two markers being studied lie on the same chromosome. If the LOD analysis establishes linkage then it can also provide a measure of the most likely recombination frequency.
THE LOD SCORE Computerized LOD score analysis is a simple way to analyze complex family pedigrees in order to determine the linkage between a trait and a marker, or two markers. The method briefly, works as follows: Establish a pedigree Make a number of estimates of recombination frequency Calculate a LOD score for each estimate The estimate with the highest LOD score will be considered the best estimate.
LOD SCORE The LOD score is calculated as follows: LOD = Z =Log10 probability of birth sequence with a given linkage probability of birth sequence with no linkage By convention, a LOD score greater than 3.0 is considered evidence for linkage. On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage.
GENETIC MAP AS A FRAMEWORK FOR PHYSICAL MAP CONSTRUCTION:
PHYSICAL MAP Determination of physical distance between two points on chromosome. Distance in base pairs Example: between physical marker and a gene. Need overlapping fragments of DNA Requires vectors that accommodate large inserts Examples: cosmids, YACs, and BACs
CONTD. Divided into 2 groups: Low Resolution Physical mapping: i. Cytogenetic map ii. cDNA map iii. Contig map High Resolution Physical mapping: i. Macrorestriction map ii. RH mapping iii. Sequence map
LOW RESOLUTION PHYSICAL MAPPING
1. CYTOGENETIC MAP Chromosomal mapping. Genes or other identifiable DNA fragments are assigned to their respective chromosome. Based on the distinctive banding patterns. Used to locate genetic markers.
2. CDNA MAP Shows the position of expressed DNA regions. Synthesized in the laboratory using mRNA as a template. Can be mapped to genomic regions. Provide the chromosomal location of the genes whose functions are currently unknown.
3. CONTIG MAPS Bottom up mapping. Involves cutting the chromosome into small pieces. Can be verified by FISH which localizes cosmids to specific regions within chromosomal bands. Consist of a linked library of small overlapping clones.
FLUORESCENT IN SITU HYBRIDIZATION (FISH) FISH is an optical mapping. FISH enables the position of a marker on a chromosome or extended DNA molecule to be directly visualized. In optical mapping the marker is a restriction site and it is visualized as a gap in an extended DNA fiber. In FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe.
FLUORESCENT IN SITU HYBRIDIZATION
HIGH RESOLUTION PHYSICAL MAPPING
1. MACRORESTRICTION MAPS Single chromosome is cut into large pieces. Depicts the order of and distance between sites at which rare- cutter enzymes cleave. Simplest way to construct is to compare the fragment sizes. The scale of restriction mapping limited by the sizes of the restriction fragments.
2. RADIATION HYBRID MAPPING Shows an estimated distance between genetic markers. A scientist exposes DNA to measure doses of radiation. Useful for ordering markers in regions where highly polymorphic genetic markers are scarce. Bridge between linkage map and sequence maps.
3. SEQUENCE MAPPING Sequence tagged site (STS) mapping. Short sequence of DNA. Exact location and order of the bases of sequence must be known. May occur only once in the chromosome.
COMMON SOURCES OF STS
I. EXPRESSED SEQUENCE TAGS(ESTS) Obtained by analysis of cDNA clones. cDNA is prepared by converting mRNA into double stranded DNA. Thought to represent the sequences of the genes being expressed.
II. SIMPLE SEQUENCE LENGTH POLYMORPHISMS(SSLPS) • Most genomes contain repeats of three or four nucleotides • Length of repeat varies due to slippage in replication • Use PCR with primers external to the repeat region • On gel, see difference in length of amplified fragment
NEED TO INTEGRATE PHYSICAL AND GENETIC MAPS: STS based mapping has its limitations. DNA fragments may lost or mistakenly mapped to a wrong position. DNA fragment may become contaminated with host genetic material. Comparing and integrating STS based physical maps with genetic, RH, cytogenic maps.
CONTD Ultimate objective of human genome was to complete DNA sequence for the organism. In order to locate genes and other interesting features. So in order to master sequence of chromosome involves several sequencing method: 1. Sequence assembly by clone Contig method. 2. Whole genome Shotgun sequencing.
1. SEQUENCE ASSEMBLY BY THE CLONE CONTIG METHOD: Conventional method for obtaining sequence of a eukaryotic genome. Genomes are broken into fragments of upto 1.5 Mb in length. Built up by identifying clones containing overlapping fragments.
2. WHOLE GENOME SHOTGUN SEQUENCING Uses a map to aid assembly of the master sequence Used to speed up the acquisition of contig sequence data for large genomes such as human genome. At least two libraries are used.
MAPPING PHASE OF THE HUMAN GENOME PROJECT Discovery of RFLPs. In 1987 first human RFLP map was published. Goal was a genetic map with density of one marker per 1 Mb. The 1994 map contained 5800 markers of which over 4000 were SSLPs.
SEQUENCING THE HUMAN GENOME The whole genome shotgun was first proposed as an alternative to contig method. The first draft sequence of an entire human chromosome (22) was published in December 1991. Finally on June 26, 2000 Francis Collins and Craig Venter jointly announced the completion of project.
HUMAN GENOME Contains about 20,000 to 30,000 genes. Only 1-2% is coding region. Rest are “junk DNA”. Some sections of the human genome have a sequence almost exactly the same as equivalent sections in other vertebrates .
FUTURE OF THE HUMAN GENOME PROJECT Completion of a finished sequence is not only the goal. Use of comparative genomics. Direct development of new drugs and therapies against cancer and other diseases.
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