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Chapter7 Analyzing DNA ＆ gene structure, variation ＆ expression. §1 sequencing ＆ genotyping DNA §2 Identifying genes in cloned DNA ＆ establishing their structure §3 Studying gene expression. Ⅰ.sequencing ＆ genotyping DNA.
◆The Enzymatic method, in which the sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains, these chains terminating at specific nucleotide positions
(Sanger Method / chain terminator sequencing)
Chain termination DNA sequencing is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another by polyacrylamide gel electrophoresis .This means that it is possible to resolve a family of molecules, representing all lengths from 10 to 1500 nucleotides, into a series of bands
The polymerase enzyme does not discriminate between dNTPs and ddNTPs, so the dideoxynucleotide can be incorporated into the growing chain, but it then blocks further elongation because it lacks the 3-hydroxyl group needed to form a connection with the next nucleotide
ddCTP why we use ddCTP?
the primer is needed because template-dependent DNA polymerases cannot initiate DNA synthesis on a molecule that is entirely single-stranded: there must be a short double-stranded region to provide a 3 - end onto which the enzyme can add new nucleotides.
The resulting DNA will be double stranded so cannot be used directly in sequencing. Instead, it must be converted into single-stranded DNA by denaturation with alkali or by boiling.
shortcoming :it can be difficult to prepare plasmid DNA that is not contaminated with small quantities of bacterial DNA and RNA, which can act as spurious templates or primers in the DNA sequencing experiment.
Obtaining single-stranded DNA by cloning in a bacteriophage M13 vector
Three criterion in particular must be fulfilled by a sequencing enzyme:
must have high processivity so that it does not dissociate from the template before incorporating a chain-terminating nucleotide.
※desirable the polymerase does not remove the chain termination nucleotide once it has been incorporated.
Kelenow enzyme; Taq polymerase; sequenase;
Automated DNA sequencing using fluorescent primers
a technique for detecting sequences within a cloned genomic DNA that are capable of splicing to exons within a specialized vector.
a hybridization-based method for retrieving genomic clones that have counterparts in a cDNA library
cognate cDNAs corresponding to genes found within the YAC will bind preferentially to the YAC DNA.
*YAC: yeast artificial chromosome
early approaches used immobilized YACs and NOW used solution hybridization reaction and
biotin- streptavidin capture methods
A PCR-based technique for mapping the end of an RNA molecule.
Nuclease S1 protection and primer extension assay
Primer extension is used to map the 5' ends of DNA or RNA fragments. It is done by annealing a specific oligonucleotide primer to a position downstream of that 5' end. The primer is labeled, usually at its 5' end, with 32P. This is extended with reverse transcriptase, which can copy either an RNA or a DNA template, making a fragment that ends at the 5' end of the template molecule. DNA polymerase can also be used with DNA templates.
The 5`- or 3`-end of a transcript can be identified by hybridization a longer, end-labeled antisense fragment to the RNA. The hybrid is treated with nuclease S1 to remove single-stranded regions, and the remaining fragment`s size is measured on a gel.
Gene expression screening
RNA transcripts protein
1.Northern blot hybridization
2.Tissue in situ hybridization
3.Whole mount in situ hybridization
2.m RNA differential display
This approach affords low resolution expression patterns by hybridizing a gene or cDNA probe to total RNA or poly (A)+ RNA extracts prepared from different tissues or cell lines. Because the RNA is size-fractionated on a gel, it is possible to estimate the size of transcripts. The presence of multiple hybridization bands in one lane may indicate the presence of differently sized isoforms.
In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence in a portion or section of tissue.
Whole mount in situ hybridization
an extension of tissue ISH is to study expression in a whole embryo.
and whole mount ISH is a popular methods for tracking expression during development in whole embryos from vertebrate organisms.
for single cell expression profiling
FISH is a cytogenetic technique which can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. It uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity.
※large-scale expression screening using microarrays
a PCR-based technique for comparing the mRNA species that are expressed in two related sources of cells to pick out differentially expressed genes.
the primary antibody is used as an intermediate molecule and is not linked directly to a labeled group. Once bound to its target, the primary antibody is in turn bound by a secondary reagent which is conjugated to a reporter which may be a fluorochrome, an enzyme or colloidal gold.
a method to detect protein in a given sample of tissue homogenate or extract.
first uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane where they are "probed" using antibodies specific to the protein. As a result, we can examine the amount of protein in a given sample and compare levels between several groups.
Immunohistochemistry refers to the process of localizing proteins in cells of a tissue section exploiting the principle of Abs binding specifically to Ags in biological tissues.
Sometimes used to screen RNA expression
This method is used when investigating the subcellular location for a protein of interest. A suitable fluorescent dye, such as fluorescein or rhodamine is coupled to the desired antibody, enabling the relevant protein to be localized within the cell by fluorescence microscopy
Higher resolution still of the intracellular localization of a gene product or other molecule is possible using electron microscopy. The antibody is typically labeled with an electron-dense particle, such as colloidal gold spheres.
the GFP gene is frequently used as a reporter gene
when the GFP gene was cloned and transfected into target cells in culture, expression of GFP in heterologous cells was also marked by emission of the green fluorescent light.