Genomics
Genomics involve genome sequence, genome organization, genome function and genome evolution.
Structural Genomics: genome structure, sequence, gene location, genome organization
Functional Genomics: gene function and expression
Genome mapping
Genetic mapping
RFLP
AFLP
RAPD
SSLP
SNP
Genome mapping
Physical mapping
Chromosome walking
Fingerprinting using restriction enzyme
Sequenced-tagged sites
Radiation hybrid cells
RFLP (Restriction Fragment Length Polymorphism)
AFLP-PCR or just AFLP is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990s.
AFLP uses restriction enzymes (MseI and EcoRI) to digest genomic DNA,
followed by ligation of adaptors to the sticky ends of the restriction fragments.
A subset of the restriction fragments is then selected to be amplified. This selection is achieved by using primers complementary to the adaptor sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments.
The amplified fragments are separated and visualized on denaturing polyacrylamide gels, either through autoradiography or fluorescence methodologies, or via automated capillary sequencing instruments.
The resulting data are not scored as length polymorphisms, but instead as presence-absence polymorphisms.
AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA.
AFLP (Amplified Fragment Length Polymorphism)
AFLP
procedure
This image of an AFLP (Amplified Fragment Length Polymorphism) assay is an example of a genetic fingerprinting method that is used to assess migratory connectivity.
Analysis of AFLP® assay data from the P1, P2, and F1 samples. Plots on the left contain the AFLP® peaks (blue) and size standard peaks (red) for the three samples. The plot on the right side shows the bins (gray bars) and allele calls.
RAPD: Random Amplification of Polymorphic DNA.
It is a type of PCR reaction, but the segments of DNA that are amplified are random.
Several arbitrary, short primers (8–12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify.
By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.
No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where.
This makes the method popular for comparing the DNA of biological systems with less attention or in a system in which relatively few DNA sequences are compared
RAPD is not suitable for forming a DNA databank, relies on a large, intact DNA template, limited in the use of degraded DNA samples. The phylogeny of diverse plant and animal species.
RAPD
Procedure
Individual #1
Individual #2
RAPD
SSLP (Simple Sequence Length Polymorphism)
Variance in the length of SSLPs (repeated sequences) can be used to understand genetic variance between two individuals in a certain species.
Minisatellite (VNTR:variable number of tandem repeats, 10-100 nt): These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.
Individual repeats can be removed from (or added to) the VNTR via recombination or replication errors, leading to alleles with different numbers of repeats. Flanking the repeats are segments of non-repetitive sequence, allowing the VNTR blocks to be extracted with restriction enzymes and analyzed by RFLP, or amplified by the polymerase chain reaction (PCR) technique and their size determined by gel electrophoresis.
Identity Matching- both VNTR alleles from a specific location must match. If two samples are from the same individual, they must show the same allele pattern.
Inheritance Matching- the VNTR alleles must follow the rules of inheritance. In matching an individual with his parents or children, a person must have an allele that matches one from each parent. If the relationship is more distant, such as a grandparent or sibling, then matches must be consistent with the degree of relatedness.
Large number of cells (10,000) is needed so this method is more useful for paternity tests than forensic tests.
VNTR
VNTR
Microsattelite (STR: simple tandem repeats) , 1-13 nt):
Microsatellites can be amplified for identification by PCR process, using the unique sequences of flanking regions as primers. This process results in production of enough DNA to be visible on agarose or polyacrylamide gels; only small amounts of DNA are needed. Primers that flank microsatellite loci are simple and quick to use, but the development of correctly functioning primers is often a tedious and costly process.
Advantage: Easier amplification using PCR, more common in the genome compared to VNTRs
SNP (Single Neucleotid Polymorphism)
Single-nucleotide polymorphisms may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions (regions between genes).
SNPs within a coding sequence can be synonymous and nonsynonymous SNPs. Synonymous SNPs does not affect the protein sequence while nonsynonymous SNPs change the amino acid sequence of protein. The nonsynonymous SNPs are of two types: missense and nonsense.
SNPs that are not in protein-coding regions may still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is referred to as an eSNP (expression SNP) and may be upstream or downstream from the gene.
These genetic variations between individuals (particularly in non-coding parts of the genome) are exploited in DNA fingerprinting, which is used in forensic science .
These genetic variations underlie differences in our susceptibility to disease. The severity of illness and the way our body responds to treatments are also manifestations of genetic variations. For example, a single base mutation in the Apolipoprotein E gene is associated with a higher risk for Alzheimer's disease.
Arrayit methods provide universal microarray-based platforms for SNP genotyping. In one VIP approach, specific chromosomal loci are amplified by use of the polymerase chain reaction (PCR), printed into microarrays, and hybridized with fluorescent oligonucleotides. The fluorescent microarrays are then scanned for fluorescence emission and signal strengths provide genotyping information.
Chromosome Walking
The first thing you need to do is create two genomic libraries of the same DNA but each library used a different restriction enzyme, such as EcoR I and Sal I.
Next, you must have a probe that is linked to your favorite gene (YFG). In this example, we want to clone the cystic fibrosis gene (CF). You would screen a genomic library with this probe and isolate a piece of DNA that binds to your probe. If you cloned an EcoR I restriction fragment from the genomic library (let's say it is fragment #3 of many possible fragments) that binds to a particular probe (called MET) that is linked to CF. You want to slide down the chromosme from MET towards CF so you can clone and sequence CF. D7S8 is another RFLP marker located on the other side of CF so CF is located between MET and D7S8.
To clone CF, you will employ chromosomal walking to take baby steps towards CF, starting with the EcoR I restriction fragment #3 you just cloned. Now, you need to generate a restriction map of EcoR I fragment #3. You must digest the Eco RI restriction fragment with multiple restriction enzymes and analyse the results on an agarose gel as shown here:
If you then performed a Southern blot with this gel and used the original MET probe that allowed you to isolate the EcoR I restricion fragment, you might see the following resutls on an X-ray film:
The next task at hand is to isolate the 2.5 kb Sal I – EcoR I fragment and use it as your second probe on the Sal I genomic library because this 2.5 kb piece is the DNA fragment furthest from the MET marker and therefore must be closer to CF. You are ready to screen the Sal I genomic library that used identical DNA but was digested with the restriction enzyme Sal I instead of EcoR I. Because probe #2 is flanked on the left by a Sal I site, you know any new fragment that has Sal sites on both ends and binds to the second probe will extend towards the right (in the direction of CF) as shown:
When you have cloned a Sal I fragment that binds to probe #2, you need to figure out its restriction map the same way we did for the EcoRI fragment #3 above. This process continues until you reach D7S8. The final product pf a chromosomal walk is a series of overlapping restriction maps starting at your original probe (MET) and extending to D7S8. The final combined restriction map, and the overlapping fragments, might look like this:
http://www.bio.davidson.edu/courses/genomics/method/chromwalk.html
The Sequence-Tagged Site (STS) :
A relatively short, easily PCR-amplified sequence (200 to 500 bp) which can be specifically amplified by PCR and detected in the presence of all other genomic sequences and whose location in the genome is mapped.
Single-copy DNA sequences of known map location could serve as markers for genetic and physical mapping of genes along the chromosome.
The advantage of STSs over other mapping landmarks is that the means of testing for the presence of a particular STS can be completely described as information in a database: anyone who wishes to make copies of the marker would simply look up the STS in the database, synthesize the specified primers, and run the PCR under specified conditions to amplify the STS from genomic DNA.
STS-based PCR produces a simple and reproducible pattern on agarose or polyacrylamide gel. In most cases STS markers are co-dominant, i. e., allow hetorozygotes to be distinguished from the two homozygotes.
The DNA sequence of an STS may contain repetitive elements, sequences that appear elsewhere in the genome, but as long as the sequences at both ends of the site are unique and conserved, researches can uniquely identify this portion of genome using tools usually present in any laboratory.
Thus, in broad sense STS include such markers as microsatellites (SSRs, STMS or SSRPs), SCARs, CAPs, and ISSRs.