Construction of human gene map through map integration- from genetic map to physical map to sequence map

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Published on September 16, 2014

Author: preetysweta22

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Construction of human gene map through map integration- from genetic map to physical map to sequence map

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.

DNA MARKER

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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