The document provides an overview of various molecular biology techniques used in nutrition research, including:
1) Purification and handling of DNA/RNA, gel electrophoresis, nucleic acid hybridization, cutting and joining DNA using restriction enzymes and ligases.
2) Methods for introducing DNA into cells including plasmids, viruses, and polymerase chain reaction (PCR).
3) DNA sequencing using dideoxynucleotides, electrophoresis to resolve sequenced fragments, and decoding the sequence data.
olymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.
olymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.
In this slide briefly describe some important note on pcr,rapd,and aflp,which helps to understand the students about this normally .
I wish for your future goal that you will shine one day inshallah .
Thank you for watching
Guide to Molecular Cloning - Download the GuideQIAGEN
Molecular cloning can be sometimes tricky with significant challenges involved. Overcome the challenges with the essential knowledge and tips for successful cloning.
Rapid and real-time diagnosis of Laem-Singh virus using a portable Real-Amp T...Narong Arunrut
The most prominent of growth-retarded in black tiger shrimp (Penaeus monodon) is associated with an infection of Laem-Singh virus (LSNV) that has been involved the reduction of annual production volume. To more accurately, rapidly and easily determine its effect on shrimp industries for further control disease, a portable Real-Amp Turbidimeter with real-time detection method was evaluated for LSNV detection.The device exploited the turbidity that produced by-product of magnesium pyrophosphate and displayed turbidity values in real-time on the provided software screen. The results of quantitative can be accomplished by reaction time measuring. The sensitivity of a turbidity detection was comparable to that of the nested RT-PCR, while it was 1000-time more sensitive than RT-PCR. With the use of a portable Real-Amp Turbidimeter. The application of RT-LAMP using a portable Real-Amp Turbidimeter offers a simple and rapid detection platform for LSNV detection in the field assessment. So, This 's awesome technic not only for detection of LSNV but also for many pathogen.
In this slide briefly describe some important note on pcr,rapd,and aflp,which helps to understand the students about this normally .
I wish for your future goal that you will shine one day inshallah .
Thank you for watching
Guide to Molecular Cloning - Download the GuideQIAGEN
Molecular cloning can be sometimes tricky with significant challenges involved. Overcome the challenges with the essential knowledge and tips for successful cloning.
Rapid and real-time diagnosis of Laem-Singh virus using a portable Real-Amp T...Narong Arunrut
The most prominent of growth-retarded in black tiger shrimp (Penaeus monodon) is associated with an infection of Laem-Singh virus (LSNV) that has been involved the reduction of annual production volume. To more accurately, rapidly and easily determine its effect on shrimp industries for further control disease, a portable Real-Amp Turbidimeter with real-time detection method was evaluated for LSNV detection.The device exploited the turbidity that produced by-product of magnesium pyrophosphate and displayed turbidity values in real-time on the provided software screen. The results of quantitative can be accomplished by reaction time measuring. The sensitivity of a turbidity detection was comparable to that of the nested RT-PCR, while it was 1000-time more sensitive than RT-PCR. With the use of a portable Real-Amp Turbidimeter. The application of RT-LAMP using a portable Real-Amp Turbidimeter offers a simple and rapid detection platform for LSNV detection in the field assessment. So, This 's awesome technic not only for detection of LSNV but also for many pathogen.
Nutrition and Exercise: Foods to Eat After You WorkoutBruce Strebinger
Nutrition and exercise go together like peanut butter and jelly, which is actually one great thing to put into your body after you workout. But what are some other foods that can help you maximize your gains?
My recent introduction talk for the Nutrigenomics Masterclass 2011in Wageningen (The Netherlands):
How to use Nutrigenomics & molecular nutrition? From challenges to solutions
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
PCR is a method for amplifying a specific sequence of DNA from a complex mixture, without the lengthy process of cloning.
It does require the knowledge of some DNA sequence information which flanks the fragment of DNA to be amplified (target DNA).
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
this ppt contain about pcr technique and its three process,primers in pcr,dna polymerase in pcr,melting temp of dna in pcr and applications of pcr technology
7. Agarose Gel Preparation Agarose : fine white powder; polysaccharide (galactose polymer) isolated from seaweed. 1% (w/v) dissolves in Tris-acetate buffer at ~60 ° C and the solution sets at ~30 ° C
14. HYBRIDISATION OVEN Incubate filter and probe in hybridisation buffer TREAT and BLOT GEL Transfer to nylon membrane nylon membrane and transferred DNA Southern/Northern Blotting and Hybridisation
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18. Ligation of DNA Eco R I OH 3’ 5’ PO 4 T4 DNA ligase catalyses the formation of phosphodiester bonds PO 4 5’ 3’ OH T4 DNA Ligase T4 DNA Ligase Stu I Circular DNA Eco R I G CTTAA AATTC G
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21. A General Laboratory Plasmid Multiple Cloning Site A foreign gene can be ligated into a plasmid, and the genetically engineered plasmid introduced into E. coli .
22. Cloning DNA into a Plasmid Both plasmid and foreign DNA have sticky Eco R I ends Insertion into E. coli (transformation) Agar plates contain antibiotic. Grow at 37 °C Place 1 colony in liquid media + antibiotic. Grow at 37 °C Purify Plasmid DNA (Billions of copies)
23. DNA and Retroviruses can serve as vehicles for the introduction of new DNA into a cell
24. DNA / RNA viruses as ‘vehicles’ Chromosomal DNA Viral DNA Integration into genome gene x Gene Therapy and Transgenics
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27. A Thermocycler This thermocycler can accept 1500 reactions at a time, and complete them in 2 to 4 hours.
28. Principal of PCR A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 5 ’ 3 ’ 5 ’ 3 ’ A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 5 ’ 3 ’ | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T o o l t o 5 5 ° C Cool. This allows specific ‘ primers’ to anneal as shown DNA (Double Stranded) Heat Denature (Becomes Single Stranded) Heat to 72 °C. Heat resistant DNA polymerase extends new DNA from the primers Heat to 72 °C C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 3 ’ 5 ’ 3 ’ 5 ’ A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | G T A T G 5 ’ 3 ’ G T T G C | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 3 ’ 5 ’ 3 ’ 5 ’ H e a t 9 5 ° C ( D e n a t u r e s ) A d d S p e c i f i c P r i m e r s C
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30. Dideoxynucleotides ddNTPs have no 3’ OH, so when added they cannot form the phosphodiester bond required to add the next nucleotide