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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.
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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 • Chemical degradation method for DNA sequencing (Maxam ＆Gillbert,1977) • Enzymatic method for DNA sequencing (Fred Sauger,1977) ◆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
Enzymatic method for DNA sequencing (Sanger Method / chain terminator sequencing) principle : 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? dCTP
We need the primer when sequencing. why? 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. • Chain termination sequencing requires a single-stranded DNA templateThe template for a chain termination experiment is a single-stranded version of the DNA molecule to be sequenced.
The DNA can be cloned in a plasmid vector 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.
The DNA can be cloned in a bacteriophage M13 vector. Obtaining single-stranded DNA by cloning in a bacteriophage M13 vector
polymerase Three criterion in particular must be fulfilled by a sequencing enzyme: • High processivity must have high processivity so that it does not dissociate from the template before incorporating a chain-terminating nucleotide. • Negligible or zero 5 → 3 exonuclease activity • Negligible or zero 3 →5 exonuclease activity ※desirable the polymerase does not remove the chain termination nucleotide once it has been incorporated. Kelenow enzyme; Taq polymerase; sequenase;
two advantages over traditional chain termination sequencing • very little template DNA is needed, so the DNA does not have to be cloned before being sequenced. • uses double-stranded rather than single-stranded DNA as the starting material. cycle sequencing
Automated DNA sequencing Automated DNA sequencing using fluorescent primers
This method uses fluorescence labeling, the DNA labeled by incorporating a primer or dNTP which carries a fluorescence. Unlike conventional DNA sequencing, this method use of different fluorescence in the 4 base, and all 4 reactions can be loaded into a single lane. During electrophoresis, a monitor detects and records the fluorescence signal as the DNA passes through a fixed point in the gel . The output is in the form of intensity profiles for each of the differently colored fluorophores , but the information is simultaneously stored electronically. This precludes transcription errors when an interpreted sequence is typed by hand into a computer file.
Ⅱ. Indentifying genes is cloned DNA ＆establishing their structure • Exon trapping a technique for detecting sequences within a cloned genomic DNA that are capable of splicing to exons within a specialized vector.
This requires a special type of vector that contains a minigene consisting of two exons flanking an intron sequence, the first exon being preceded by the sequence signals needed to initiate transcription in a eukaryotic cell .To use the vector the piece of DNA to be studied is inserted into a restriction site located within the vector's intron region. The vector is then introduced into a suitable eukaryotic cell line, where it is transcribed and the RNA produced from it is spliced. The result is that any exon contained in the genomic fragment becomes attached between the upstream and downstream exons from the minigene. RT-PCR with primers annealing within the two minigene exons is now used to amplify a DNA fragment, which is sequenced. As the minigene sequence is already known, the nucleotide positions at which the inserted exon starts and ends can be determined, precisely delineating this exon.
cDNA selection/capture a hybridization-based method for retrieving genomic clones that have counterparts in a cDNA library principle : 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
RACE-PCR(rapid amplification of cDNA ends-PCR) A PCR-based technique for mapping the end of an RNA molecule.
In the simplest form of this method one of the primers is specific for an internal region close to the beginning of the gene being studied. This primer attaches to the mRNA for the gene and directs the first reverse-transcriptase-catalyzed stage of the process, during which a cDNA corresponding to the start of the mRNA is made . Because only a small segment of the mRNA is being copied, the expectation is that the cDNA synthesis will not terminate prematurely, so one end of the cDNA will correspond exactly with the start of the mRNA. Once the cDNA has been made, a short poly(A) tail is attached to its 3 - end. The second primer anneals to this poly(A) sequence and, during the first round of the normal PCR, converts the single-stranded cDNA into a double-stranded molecule, which is subsequently amplified as the PCR proceeds.
Mapping transcription start and sites and defining exon-intron boundaries Nuclease S1 protection and primer extension assay
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.
Nuclease S1 protection 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.
Ⅲ.Studying gene expression Gene expression screening ↓ Target ↙ ↘ RNA transcripts protein ↓ ↓ Hybridization antibody
gene-based expression analyses: hybridization analyses 1.Northern blot hybridization 2.Tissue in situ hybridization 3.Whole mount in situ hybridization PCR analyses 1.RT-PCR 2.m RNA differential display
Ⅱ.protein-based expression analyses: • Immunoblotting (Western blotting) • Immunocytochemistry →immunohistochemistry • Immunofluorescence microscopy • Ultrastructural studies
Northern blot hybridization 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.
Tissue in situ hybridization 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.
※fluorescence in situ hybridization (FISH) 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
mRNA differential display 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 method uses a modified oligo(dT) primer which has a different single nucleotide or dinucleotide at the 3- end causing it to bind to the poly(A) tail of a subset of mRNAs. For example, if the oligonucleotide TTTTTTTTTTTCA(T11CA) is used as a primer, it will preferentially prime cDNA synthesis from those mRNAs where the dinucleotide TG precedes the poly(A) tail. The second primer which is used is usually an arbitrary short sequence (often 10 nucleotides long but, because of mismatching, especially at the 5 - end, it can bind to many more sites than expected for a decamer). The resulting amplification patterns are deliberately designed to produce a complex ladder of bands when size-fractionated in a long polyacrylamide gel .
Antibody labeling and detection system 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.
Immunoblotting (Western blotting) 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 (IHC) 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
Immunofluorescence microscopy 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
Ultrastructural studies 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.
Green fluorescent protein (GFP) 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.
