gene mapping
TRANSCRIPT
CONTENTS
* PLANT CHROMOSOME ANALYSIS* MAPPING OF PROKARYOTIC &
EUKARYOTIC GENES.* USES OF PCR IN GENE MAPPING* MOLECULAR MPS-RFLP, RAPD AND
THEIR APPLICATION ON DETECTION OF ADULTERANTS
* PHYSICAL MAPS IN-SITU HYBRIDIZATION
INTRODUCTION TO PLANT CHROMOSOME ANALYSIS
• Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes.
• During the past decade, significant progress has been made in the development of methods for the preparation of plant chromosome suspensions suitable for flow cytometric analysis.
• sorted chromosome were used for the establishment of chromosome specific DNA libraries & for gene mapping.
TECHNIQUES FOR QUANTITATIVVE CHROMOSOME ANALYSIS
CYTOPHOTOMETRYCYTOFLUROMETRYIMAGE ANALYSIS
TYPES OF FLOW CHAMBER IN FLOW CYTOMETERS
PRINCIPLE USED IN FLOW SORTERS
PREPARATION OF CHROMOSOME SUSPESION• The problem associated with preparation of high quality suspension of plant
chromosomes may be considered the main reason for the delay in flow cytometric analysis and sorting of plant chromosomes,
• An ideal chromosome suspension should contain intact & well-dispersed chromosomes and should be free of contaminating cellular debris.
• This is difficult to achieve with plant material for several reasons: 1. It is not easy to obtain high degree of mitotic synchrony in plant tissues2. Chromosome stickiness and clumping is often observed after treatment with metaphase
blocking agents.3. Plant chromosome blocked in metaphase have tendency to split into single chromatids.4. The present of a rigid cell wall makes the release of chromosomes more difficult in
comparisons with human or animal calls 5. Plant chromosomes are not stable for longer periods in currently used isolation buffers.
ISOLATION OF CHROMOSOME
1. INDUCTION OF CELL CYCLE SYNCHRONY2. ACCUULATION OF CELLS IN METAPHASE3. RELEASE OF CHROMOSOMES
CHROMOSME ANALYSIS
• UNIVARIATE FLOW KARYOTYPING• BIVARIATE FLOW KARYOTYPING• With most od the commercially available flow cytometers, wo basic optical parameters of particles can be
analysed:1. Light scatter2. Fluorescence emission
• Chromosome morphology( length, shape) may vary considerably among the population of chromosomes of the same type, which is reflected by broad distribution of light scatter intensity. This does not permit discrimination of single chromosome types.
• On the other hand DNA content is essentially constant for each chromosome type, and thus fluorescence intensity related to the chromosomal DNA contents has been used as a basic parameters in flow cytometric analysis of isolated chromosome since its early beginnings (Carrano et. Aa.)
CHROMOSOME STAINING
1. INTERCALATING DYES2. DNA-SPECIFIC DYES3. FLUORESCENT ANTIBIOTICS4. DYE COMBINATIONS
ADVANTAGE & FUTURE OF FLOW CYTOMETRY
• As yet, flow cytometry is not a major technique in plant chromosome analysis. however, its advantage in term of speed, accuracy, and quality of quantitative measurement make the technique one which likely to become more widespread.
• While nuclear DNA measurement can be quickly obtained, & are of value in examining purity of plant stocks, keening for aneuploidy or polyploidy, flow karyotyping is considerably behind animal work.
• Because of quantitative measurements which can be made, flow cytometry can be used for checks and quality control of many aspects of plant genetics,. In plant breeding and seed production, the ability to qualify nuclear size and hence assess purity and ploidy is likely to be useful.
BIVARIATE FLOW KARYOTYPES OF VICIA FABA, S. LUCRETTI AND J. DOLEZEL
• A) Fluorescence pulse area (FPA) and fluorescence pulse width (FPW) are used simultaneously as parameters. Groups corresponding to six pairs of chromosomes (higher values of FPW, similar values of FPA) are resolved. Chromatid doublets of different chromosomes are shown also.
• B) Contour plot of MI/DAPI stained field bean chromosomes. The chromosomes are stained with two fluorochromes, 4',6-diamidino- 2-phenylindole (DAPI) at final concentration of 1.5 µM and mithramycin A (MI) at final concentration of 20 µM for at least 30 min. MgSO4 is added to chromosome suspension at final concentration of 10 mM prior to staining.
METAPHASES OF FEULGEN-STAINED PEA (PISUM SATIVUM) ROOT TIP CHROMOSOMES-S. LUCRETTI, G. GUALBERTI, AND J. DOLEZEL
• A) and B): metaphases of Feulgen-stained pea (Pisum sativum L.) root tip chromosomes observed by their fluorescent emission after green excitation, Standard and reconstructed karyotype L-84, respectively.
• C) and D): flow-karyotyping histograms of DAPI-stained chromosome suspensions for the Standard and L-84, respectively. Capital letters indicates chromosome specific peaks, as assigned after chromosome sorting.
FLOW-KARYOTYPING OF DNA INTEGRAL FLUORESCENCE (FPA) OF DAPI-STAINED PEA CHROMOSOMES, S. LUCRETTI,
G. GUALBERTI, J. MACAS, AND J. DOLEZEL
• Flow-karyotyping of DNA integral fluorescence (FPA) of DAPI-stained pea chromosomes. Inside pictures show sorted chromosomes from regions R1 (I+II) and R2 (VI+III and I), DAPI-stained; from regions R3 (III+IV) and R4 (V+VII) after PRINS labelling for rDNA (chromosomes IV and VII with secondary constriction are labelled)
MAPPING OF EUKARYOTIC & PROKARYOTIC GENES
MAPPING OF PROKARYOTIC GENES• Gene information is processed differently in prokaryotes & eukaryotes.
Because prokaryotes lack nuclear envelope, messenger RNA (mRNA) can associate with ribosome in the cytoplasm as the mRNA is being formed.
• The following features are important for gene recognition: (1) ORF length(2) presence of a ribosome binding site (RBS) upstream of the start codon(3) specific pattern of codon usage that is different from triplet frequencies in
non-coding regions (‘coding potential’), as well as other similar statistical parameters; and
(4) similarity to known genes.
Intrinsic approach to gene recognitionIntrinsic, or ab initio, approaches use the first three types of data. Hidden Markov models (HMM) provide a convenient language for integrating these diverse parameters of candidate genes in genomic sequences. Extrinsic methods rely on the comparative analysis of genomic DNA sequences using alignment with known genes and proteins. Ribosome binding sitesRibosome binding sites are located in the (20) ... (1) region upstream of start codons and serve to direct ribosomes to the correct translation start position. A part of RBS is formed by the purine-rich Shine–Dalgarno (SD) sequence, which is complementary to the 39 end of the 16S rRNA. A number of early papers described methods for recognition of ribosome binding sites using statistical, pattern recognition or neural network modelling of experimentally mapped sites.There are two approaches to the recognition of ribosome binding sites in the absence of a learning sample One possibility is to rely on the universal mechanisms of RBS recognition via basepairing of the SD box
and the 39-terminus of the 16S rRNA The other possibility is to derive a ‘pseudo-learning’ sample of candidate translation initiation sites using
protein coding regions predicted by database search or statistical analysis.A convenient technique for integration of diverse parameters is the HMM. HMM24 is a Markov chain of hidden states. Each state is assigned a distribution of emission probabilities (Bernoulli or Markov) that generate the observed nucleotide sequence.
Extrinsic approachesExtrinsic analysis involves sequence similarity searches. Candidate gene products are searched against protein sequence databanks. BLASTX, the most popular program of this class, performs six-frame translation of the query DNA and compares the resulting amino acid sequences to known proteins.The simplest way to combine the extrinsic and intrinsic approaches is to apply them in parallel. MATERIALS AND METHODS The prediction is done in three steps: • Building the tables of orthologues. • Applying a dynamic programming algorithm to align pairs of orthologous genes. • Filtering of results and identification of suspicious gene starts and possible frame-shifts.
MAPPING OF EUKARYOTIC GENES• The genomes of eukaryotic organisms contain hundreds to thousands of
genes (an estimated 30,000-50,000 in humans). Yet there are only a handful of chromosomes. Thus, each chromosome in a eukaryotic genome must contain a large number of genes.
• The transmission of genes located on the same chromosome may violate Mendel’s Law of Independent Assortment, particularly if they are located very close together along the same arm of a chromosome.
• This set of lecture notes will explain why, and provide the theoretical basis for mapping genes along a chromosome by following the degree to which they violate Mendel’s Law of Independent Assortment during genetic crosses.
Linkage and Crossing Over• In eukaryotic species, each linear chromosome contains a long piece of DNA• A typical chromosome contains many hundred or even a few thousand different genes• The term “linkage” has two related meanings
• 1. Two or more genes can be located on the same chromosome• 2. Genes that are close together tend to be transmitted as a unit
• Chromosomes are called linkage groups• They contain a group of genes that are linked together
• The number of linkage groups is the number of types of chromosomes of the species• For example, in humans
• 22 autosomal linkage groups• An X chromosome linkage group• A Y chromosome linkage group
• Genes that are far apart on the same chromosome may independently assort from each other due to crossing-over during meiosis.
• Occurs during prophase I of meiosis • Homologous chromosomes exchange DNA segments
LINKAGE GROUPSChromosomes are called linkage groups – They contain a group of genes that are linked together They contain a group of genes that are linked together.• The number of linkage groups is the number of types of chromosomes of the
species – For example, in humans " 22 autosomal linkage groups “ An X chromosome linkage group " A Y chromosome linkage group• Genes that are far apart on the same chromosome can independently assort
from each other • – This is due to This is due to crossing-over or recombination rossing-over or
recombination
PCR IN GENE MAPPING• The polymerase chain reaction (PCR)’ is an in vitro method of nucleic acid synthesis that
enables the specific replication of a targeted segment of DNA, providing a rapid, highly sensitive, and specific means of nucleic acid detection and isolation.
• The PCR technique uses two oligonucleotide primers that complement opposite ends of each strand of a target sequence, and are oriented in such a way that DNA synthesis proceeds across the region between the primers.
• PCR was originally performed using Klenow fragment as the DNA polymerase, and thermocycling was accomplished by transferring reaction tubes between a series of water baths set at different temperatures. The procedure required adding new enzyme after each cycle, and significant manipulation of reaction tubes. PCR has since been improved through the use of thermostable Taq DNA polymerase and automated instrumentation for thermal cycling. These ad- vances eliminated the need to add fresh enzyme after each cycle, as well as the need for manual transfer of tubes previously required to achieve heating and cooling. I
USES OF PCR IN GENE MAPPING
• Cloning a gene encoding a known protein: primers can be designed from the sequence of amino acids or gene sequence. amplified product can be used as a probe to pull out the full length gene from a cDNA or a genomic libraries.
• Amplification of old DNA: amplifying DNA sequence from museum material or fossils to look at evolution of gene sequence( molecular evolution studies).
• Amplification of cloned DNA from vectors: convenient way of checking the inserts is to amplify DNA & analysed it by southern blotting.
• Creating mutation in cloned DNA
USES OF PCR IN GENE MAPPING
• Rapid amplification of cDNA ends (RACE): most cloned in cDNA libraries are not of full length. RACE enables 5’ or 3’ end of a transcript to be cloned as an alternative to rescreening libraries for overlapping clones. Only one gene specific primer is needed.
• Detection of bacterial & viral infection • Detection of cancer : PCR technique is used for detecting mutation that occur in
cancer & monitoring cancer therapy• Genetic diagnosis : PCR technique is also used in diagnosing inherited disorder
like cystic fibrosis, muscular dystrophy, haemophilia A & B & sickle cell anaemia.
MOLECULAR MAPS-RFLP, RAPD & THEIR APLLICATION FOR DETECTION OF
ADULTERANTS• Linkage maps of many plant species were limited in size until the advent of molecular mapping. The primary difficulty with
developing linkage maps was the inability to incorporate many markers into a single stock to be used for genetic analysis. This inability occurred because of the deleterious effects of the expression of all mutant phenotypes in the single stock. Because normal DNA or protein molecules are used to score the genetic material, molecular markers are phenotypically neutral. This is a significant advantage compared to traditional phenotypic markers.
• The three most common types of markers used today are RFLP, RAPD and isozymes. Of the three marker types, RFLPs have been used the most extensively. RFLP markers have several advantages in comparison with the RAPD and isozyme markers:
1) they are codominant and unaffected by the environment; 2) any source DNA can be used for the analysis; and 3) many markers can be mapped in a population that is not stressed by the effects of phenotypic mutations. The primary drawback to RAPD markers is that they are dominant and do not permit the scoring of heterozygous individuals. The weakness of isozyme markers is that each of the proteins that are being scored may not be expressed in the same tissue and at the same time in development. Therefore several samplings of the genetic population need to be made.
• RFLP - Restriction Fragment Length Polymorphism; a molecular marker based on the differential hybridization of cloned DNA to DNA fragments in a sample of restriction enzyme digested DNAs; the marker is specific to a single clone/restriction enzyme combination.
• RAPD - Randomly Amplified Polymorphic DNA; a molecular marker based on the differential PCR amplification of a sample of DNAs from short oligonucleotide sequences.
• AFLP - Amplified Fragment Length Polymorphism; a molecular marker generated by a combination of restriction digestion and PCR amplification.
• Isozyme - a molecular marker system based on the staining of proteins with identical function, but different electrophoretic mobility.
RFLP Loci• RFLP analysis is an application of the Southern hybridization procedure. The general
principles will be explained here and we will then discuss several papers to obtain a more in depth understanding of the procedure.
RAPD Loci• RAPD markers have recently caught the fancy of many individuals in the field of
applied plant breeding. This molecular marker is based on the PCR amplification of random locations in the genome of the plant. With this technique, a single oligonucleotide is used to prime the amplification of genomic DNA. Because these primers are 10 nucleotides long, they have the possibility of annealing at a number of locations in the genome. For amplification products to occur, the binding must be to inverted repeats sequences generally 150-4000 base pairs apart. The number of amplification products is directly related to the number and orientation of the sequences that are complementary to the primer in the genome
PHYSCAL MAPS IN-SITU HYBRIDIZATION
• In contrast to a linkage map, which specifies statistical distances between variable DNA markers and genes in terms of recombination fractions (see “Classical Linkage Mapping”), a physical map specifies physical distances between landmarks on the DNA molecule of each chromosome.
1)Low-Resolution Physical Mapping by In-Situ Hybridization2) High-Resolution Physical Mapping by Construction of Contig Maps of Overlapping Clones
• The figure at right is a schematic of a contig map for one chromosome. Right now, the top priority of the Human Genome Project is to construct a contig map for each of the twenty-four different chromosomes in the human genome. Those maps, when integrated with the corresponding genetic-linkage maps, will provide a means of finding the segments of DNA that contain disease genes (see “Modern Linkage Mapping”). The clones that make up the map also provide the material needed to sequence the human genome.
LOW-RESOLUTION PHYSICAL MAPPING BY IN-SITU HYBRIDIZATION
• One standard low-resolution method for finding the physical position of a cloned fragment is in-situ hybridization on metaphase chromosomes. We first find a segment within the cloned region whose base sequence occurs nowhere else in the genome, We then synthesize many copies of a single strand of that unique segment and label each copy with a fluorescent tag to make it useful as a DNA probe. A solution containing the DNA probe is then applied to a spread of chromosomes that have been arrested at metaphase and fixed to a microscope slide. (Metaphase is the phase of cell division during which chromosomes have condensed to form the wormlike shapes easily visible under a light microscope.) Under appropriate conditions the probe binds, or hybridizes, only to the chromosomal DNA with a base sequence exactly complementary to that of the probe (see “Hybridization” in “Understanding Inheritance”). The position on a metaphase chromosome where the probe has hybridized is imaged with a fluorescence microscope as a bright spot. Because DNA molecules are wound very tightly during metaphase, the resolution achieved with in-situ hybridization is low, about 3 million base pairs. In other words, the hybridization signals from two probes less than 3 million base pairs apart will overlap one another and cannot be resolved into two distinct spots. In-situ hybridization using four cloned inserts as probes produced the bright spots on the metaphase chromosomes in the micrograph shown on the page opposite.
HIGH-RESOLUTION PHYSICAL MAPPING BY CONSTRUCTION OF CONTIG MAPS OF
OVERLAPPING CLONES• To determine the positions of genomic landmarks with much greater resolution, we can replace the
chromosomes themselves with twenty-four contig maps, one for each of our twenty-two homologous chromosome pairs and one for each of our two sex chromosomes. A contig map is a set of contiguous overlapping cloned fragments that have been positioned relative to one another. In a complete contig map for a human chromosome, the cloned fragments would include all the DNA present in the chromosome and follow the same order found on the DNA molecule of the chromosome. As in any physical map, distances are measured in base pairs. Using these contig maps, we can localize any cloned fragment or other DNA probe, again by hybridization, to a much smaller portion of the genome, namely to one of the cloned fragments in one of the maps. Moreover, we can determine the position of any DNA probe relative to all other landmarks that have been similarly localized. Once contig maps are constructed, the entire genome will be available as cloned fragments, and we will be able to use these clones to analyze any region down to the level of its base sequence.
REFERENCES
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