(1) Maxam-Gilbert 法： 以四種化學反應分別對四種鹼基作用，每一反應只對單一種鹼基進行修飾，而在該鹼基的地方斷開，得到一系列長度不同的核酸片段。 電泳可依照這些 DNA 片段的大小，在膠體中排開，即可依序判讀 DNA 分子上核 苷 酸的序列；比較如此四組鹼基序列電泳，即可組合成整段 DNA 。 (2) Sanger 法： 以樣本 DNA 為模板，使用 DNA polymerase 進行試管中 DNA 生合成。 四個反應中，每個反應各缺單一種核苷酸，而代以其 類似物 (analogue) ，則部分合成反應會停在該類似物的核 苷 酸處，造成各種長短不一的 DNA 片段，以電泳分離如上，即可組合判讀 DNA 的序列。 上述兩種方法，均以 32 P 標示在核酸分子上，以便顯像各不同長度的核酸片段。
This step consists of heating the reaction to a temperature of 94-95°C which is held for 1-9 minutes
It is enough to completely denature complex genomic DNA
Each cycle includes three successive steps:
Each cycle takes as little as few minutes and it usually takes fewer than 20 cycles to produce as much amplified DNA as one needs
Denaturation: One to several minutes at 94-96 C , during which the DNA is denatured into single strands
Annealing: One to several minutes at 50-65 C , during which the primers hybridize or "anneal" (by way of hydrogen bonds) to their complementary sequences on either side of the target sequence
Extention: For fragments up to 3 kb, primer extension is normally carried out at 72 C
Polymerase binds and extends a complementary DNA strand from each primer and add approximately 60 bases per second at 72C .
RT-PCR, one of the most sensitive methods for the detection and analysis of rare mRNA transcripts or other RNA present in low abundance.
RNA cannot serve as a template for PCR, so it must be first transcribed into cDNA with reverse transcriptase and the cDNA copy is then amplified.
The technique is usually initiated by mixing short (12-18 base) polymers of thymidine (oligo dT) with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand cDNA .
Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment.
The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.
Hot Start PCR significantly improve specificity, sensitivity and yield of PCR
Some components essential for polymerase activity is separated from the reaction mixture until the temperature in the tubes has exceeded the optimal primer annealing temperature usually 55-65 C ˚
Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step .
The determination or quantitation of the number of copies of a given gene achieves accurate estimation of DNA and RNA targets.
Real Time PCR
Traditional PCR has advanced from detection at the end-point of the reaction. to detection while the reaction is occurring Real-Time PCR is used.
Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated . This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.
The real time system reduces the time required for PCR amplification and analysis from hours to minutes.
Monitor amplification online and in real-time.
Quickly and accurately quantify results.
Analyze melting characteristics of PCR product.
The recent development of real time PCR clearly demonstrates many advantages over other existing method with: high accuracy, wide dynamic range , specificity , sensitivity , reduced carry over contamination and rapid accurate and simultaneous quantification of multiple samples.
How DNA Sequence Is Determined? Polyacrylamide Gel Electrophoresis T A G C T A C G DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis But these bands can’t tell us the identity of the terminal nucleotides If those band with the same terminal nucleotide can be grouped, then it is possible to read the whole sequence Juang RH (2004) BCbasics ATC 32 P AT 32 P A 32 P ATCG 32 P ATCGA 32 P ATCGAT 32 P ATCGATC 32 P ATCGATCG 32 P ATCGATCGA 32 P ATCGATCGAT 32 P
How to Obtain DNA Fragments AT ATCGAT Specific Reaction to G A Terminated Biosynthetic method Chemical method Template or Non-radioactive (invisible) Producing various fragments
Phosphodiester bond 3’ 5’ di deoxy nuceotide Terminated dd NTP Sanger’s Method: How Terminated Normal Linking Can not react Juang RH (2004) BCbasics P R P R P R P R P R P R OH 5’ 3’ 1 A 1 2 3 4 5 6 A PO 4 2- H 3’ 5’ 2 H
Gene Transferred by Plasmid Plasmid gets out and into the host cell Resistant Strain New Resistance Strain Non-resistant Strain Plasmid Enzyme Hydrolyzing Antibiotics Juang RH (2004) BCbasics Drug Resistant Gene mRNA
Target Genes Carried by Plasmid 1 plasmid 1 cell Recombinant Plasmid Transformation Target Gene Recombination Restriction Enzyme Restriction Enzyme Chromosomal DNA Target Genes DNA Recombination Transformation Host Cells
Amplification and Screening of Target Gene 1 1 cell line, 1 colony X100 X1,000 Plasmid Duplication Bacteria Duplication Plating Pick the colony containing target gene =100,000
Increasing casein content of milk increase cheese production
Lactose free milk (transgene lactase)
Resistance to bacterial infections
In vivo immunization
transgene is specific Heavy and Light chain genes which
create An production against a specific antigen
Why Express rProtein in Milk Easy to purify - few other proteins in milk Doesn’t harm transgenic animal- no change to physiology rProtein is authentically modified post-translationally Large quantities Renewable source
Sheep and Pigs PIGS PST porcine somatotropin (growth hormone) adverse effects- kidney, stomach, heart, sterility human Hemoglobin to replace whole blood transfusions SHEEP Increase wool production keratin promoter growth factor
Organ Transplant Pr Problem: Rejection Antibodies from Host bind to Donor Organ Elicits Inflammatory Response Transplanted Organ Lost Solution: Transgene in Donor for Complement-Inhibiting Protein 111
Birds and Fish Birds traditional methods can not be used because of avian embryogenesis differences no ES cells found ALV resistant chickens transgene - defective ALV genome makes viral RNA and protein but blocks assembly of viral particles Fish aquaculture transgene - growth hormone
There is consequently extensive research into >200 chicken quantitative trait loci encoding for disease susceptibility, immunology, leanness, egg production, etc. ( LIU et al. 2001 ; MARIANI et al. 2001)
Chicken DT40 cell lines are avian-leukosis-virus-induced B cell lines that exhibit a high ratio of targeted to random integration of transfected DNA constructs at homologous loci (DHAR et al. 2001 ).
A feature of DT40 cell lines, however, is that they have a high degree of chromosomal rearrangements that, to date, could not be karyotyped. (WINDING and BERCHTOLD 2001 )
Some 15,000 genes are expressed in chicken tissues involved in the regulation and development of immune responses ( http://ec.europa.eu/research/agriculture/projects_showcase01_en.htm )
The chicken"s major histocompatibility complex (MHC) haplotype has a profound influence on the resistance or susceptibility to certain pathogens ( Research Project: USING THE GENOME TO UNDERSTAND IMMUNOGENETICS OF POULTRY )