Cellular and Molecular Biology Topics
Genetic Traits
Alternative forms of a gene that can occupy a particular chromosomal sites are alleles, i.e. one specific form (sequence) of a gene at a particular place (locus) on a chromosome. We inheret one allelle of each autosomal gene from our father and one from our mother.
In an autosomal dominant genetic trait, one copy of the defective gene leads to disease. There is a 50% chance of the offspring being affected.
Autosomal recessive genetic traits must have both copies of the gene defective to cause disease. Offspring of two carriers have 25% chance of being affected, and 50% change of being a carrier.
In an X-linked recessive genetic trait, the gene is on the X chromosome. If the mother is a carrier, the son has 50% chance of being affected and daughter has 50% chance of being a carrier.
Iin a complex genetic disease an increased risk of disease is inherited, but there is no single pattern of inheritnce. There are multiple genes responsible, which may act independently or may interact with themselves or with environmental conditions. There is a significant environmental component to risk. It is not deterministic, i.e. increased risk does not mean inevitavility. .
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Back to Basics: Biochemistry
DNA Cloning
Clones are large number of identical cells that arise from a single cell. Also a large number of identical recombinant DNA molecules from such a clone. As a verb, it means to isolate a specific piece of DNA.
To create a clone, foreign DNA is inserted into a specific site in a vector by cleaving the vector with a restriction endonucleases and ligating the foreign DNA to the cut vector. Many enzymes act on DNA and can be used to duplicate, label, cut, and reseal it.
Restriction endonucleases cut at specific sequences, allowing DNA to be cut reproducibly (dissected). Many restriction endonucleases leave “sticky ends” (complementary overhangs) that can be reanneal. These are useful in cloning DNA since a fragment and a vector with the same sticky ends can easily be joined by DNA ligase. DNA polymerases copy DNA and allow incorporation of labels. They require primers. DNA ligases join two pieces of DNA, sealing nicks. Reverse transcriptases from retroviruses are special DNA polymerases that use RNA as a template to make complementaty DNA (cDNA). They require primers. The cDNA can be used by DNA polymerase to synthesize the complementary strand.
A cloning vector is a molecule that can carry a DNA sequence into a cell, maintain it there over many generations and allow propagation of specific DNA molecules to provide an unlmited amount of the DNA of interest. If the foreign DNA is cut with the same restriction endonucleases as the vector and has the same “sticky ends” the process is more efficient. Vectors need to contain a replication origin, a restriction site at which foreing DNA can be added and a selectable marker. They may have other useful features, like a secondary marker and regulatory sequences.
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Back to Basics: Biochemistry
Gene Libraries
A library of clones is a collection of many different clones each carrying a foreing DNA from a particular source of interest. The aim is to have essentially all DNA from a given tissue or all parts of the genome represented in the library by at least one clone. Either cDNA or genomic DNA may be cloned, each with different benefits and uses. cDNA is complementary DNA, a copy of a mRNA. Genomic DNA is the original DNA molecule from a nuclear gene.
cDNA libraries carry a large collection of cDNA from a given tissue. Each tissue has a unique set of mRNAs, since each express only a subset of the genes in the organism. One use of this in the Human Genome Project is to clone and sequence as many cDNAs as possible from a wide variety of cells and tissues to be used for gene mapping and gene identification. Many wre sequenced just at their 3' ends and are called expressed sequence tags (EST). ESTs are short regions of cDNA (200-300 bp) randomly sequenced and used as markers for sequences in mRNA, useful for gene mapping and gene identification.
Genomic DNA libraries carry a large collection of overlapping pieces of DNA including genes and regulatory regions as well as noncoding regions between genes. These are used in studes of gene structure and regulation, and in comprehensive analysis of the gene. With the progress of the Human Genome project, one can now use the electronic information in many databases to locate and study genes. Often this can replace the tedious process of cloning a gene. Whole genomes of several organisms are now available for analysis and design of therapeutic agents.
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Advance Topics: Molecular Biology Lab
DNA Analysis
Electrophoresis separates DNA by length. This can be extremely accurate due to the uniformity of the shape and charge of DNA molecules. Agarose gels separate DNA pieces over a wide range of sizes. Denaturing polyacrylamide gels can separate fragments that differ in length by one nucleotide and are used for DNA sequencing, analyzing microsatellites markers, etc.
Southern blotting allows the detection of specific restriction fragments by hybridization with a probe. Restriction fragments are pieces of DNA from restriction endonucleases digestion. A probe is a nucleic acid that can hybridize to target sequences and thereby be used to detect a specific target sequence. A cloned copy of a gene, an RNA, or a synthetic oligonucleotide can be used as probes. Complementary strands can hybridize, allowing the detection of specific DNA pieces in a gel. By carefully adjusting stringency in order to accept different amounts of mismatching, one specific fragment of DNA can be detected (high stringency) or related sequences can be detected (low stringency).
The polymerase chain reaction provides exponential amplification of a specific segment of DNA. The sequence flanking the desired DNA must be known to design specific primers. Repeated cycles of denaturation, addition of primers and polymerization (DNA replication) are repeated. The DNA of interest is mixed with primers, buffers, nucleotides and enzymes. DNA polymerase elongates the annealed primers. Target DNA doubles in each cycle, until it makes up most of the DNA in the tube. It can the be directly analyzed.
DNA sequencing is used to determine the complete sequence of a gene. Replication is allowed to start at a fixed position (5’ end of the primer) and it ends after a particular base is inserted i.e. the reaction is not allowed to complete (~ 2% terminate and 98% continue at each position). This produces a set of nested fragmentseach with the same known 5’ end and a different base at the 3’end. The fragments are then separated by size at a resolution of a single nucleotide, by polyacrylamide electrophoresis under denaturing conditions. The separated DNA fragments are visualized using a radioactive or flourecent label. The sequence may be read directly from the blot.
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Back to Basics: Biochemistry
Detection of Genetic Differences
Southern blotting is used to identify genes or parts of genes for genetic diagnosis. Sickle cell anemia is a recessive genetic disease caused by the precipitation of hemoglobin in which both beta chains have a single amino acid difference (a valine for a glutamate) due to a replacement of a tyramine (GTG) for an adenine (GAG). MstII is a restriction endonucleases that recognizes and cleves at CCTNAGG sequences. This sequence is found in the region encoding normal hemoglobin beta chains but not in the sickle cell hemoglobin. Detection by Southern blot follows these steps:
1- DNA extraction
2- Restriction endonuclease (MstII) digestion: cuts DNA reproducibly into millions of fragments each carrying a specific portion of the genome.
3- Gel electrophoresis: separates DNA fragments by length; shorter fragments move faster than longer fragments. Each fragment moves to its characteristic position.
4- Denature and transfer to membrane: blot is 2D replica of the gel, all fragments retaining their original relative positions
5- Hybridize with labeled probe (CTTNAGG): the complimentary probe is a previously cloned copy of the DNA segment or analogous sequence, or an RNA or oligonucleotide.
6- Wash unhybridized probe off (high stringency)
7- Autoradiography: detect specific fragment complimentary to probe. This determines the position to which the sequence of interest has migrated, therefore the length of the fragment. The only places were labeled probwe is detected are were complimentary DNA migrated. Diagnosis is by fragment length: did MstII cut or did not cut? If the DNA fragment is longer than standard, it did not cut and the test is positive.
The polymerase chain reaction provides exponential amplification of a specific segment of DNA. The sequence flanking the desired DNA must be known to design specific primers. Repeated cycles of denaturation, addition of primers and polymeriztion (DNA replication) are repeated. The DNA of interest is mixed with primers, buffers, nucleotides and enzymes. DNA polymerase elongates the annealed primers. Target DNA doubles in each cycle, until it makes up most of the DNA in the tube. It can the be directly analyzed.
PCR and allele-specific oligonucleotides (ASOs) are used in genetic diagnosis to identify specific genes. DNA is amplified by PCR, bound to membranes and then probed with an oligonucleotide exactly matching the sequence of the target allele. Hybridization and washing under conditions of high stringency allows only the exact matches to anneal. This differentiates between polymorphic alleles, for example ADH2*1 and ADH2*3.
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Advance Topics: Molecular Biology Lab
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