Cloning Overview DNA can be cloned into bacterial plasmids for research or commercial applications. The recombinant plasmids can be used as a source of.

Slides:



Advertisements
Similar presentations
Chapter 4: recombinant DNA
Advertisements

Recombinant DNA Introduction to Recombinant DNA technology
3 September, 2004 Chapter 20 Methods: Nucleic Acids.
Manipulating the Genome: DNA Cloning and Analysis 20.1 – 20.3 Lesson 4.8.
1 Review Describe the process scientists use to copy DNA Use Analogies How is genetic engineering like computer programming 2 Review What is a transgenic.
Definitions: 1. Genetic engineering- remaking genes for practical purposes 2. Recombinant DNA- DNA made from two or more different organisms 3. Restriction.
Chapter 20: Biotechnology. Essential Knowledge u 3.a.1 – DNA, and in some cases RNA, is the primary source of heritable information (20.1 & 20.2)
DNA Technology- Cloning, Libraries, and PCR 17 November, 2003 Text Chapter 20.
Chapter 20~DNA Technology & Genomics. Who am I? Recombinant DNA n Def: DNA in which genes from 2 different sources are linked n Genetic engineering:
AP Biology: Chapter 14 DNA Technologies
DNA Technology Ch. 20 Figure 20.1 An overview of how bacterial plasmids are used to clone genes.
AP Biology Ch. 20 Biotechnology.
-The methods section of the course covers chapters 21 and 22, not chapters 20 and 21 -Paper discussion on Tuesday - assignment due at the start of class.
Chapter 20 DNA Technology. DNA Cloning  Gene cloning allows scientists to work with small sections of DNA (single genes) in isolation. –Exactly what.
1 Genetics Faculty of Agriculture Instructor: Dr. Jihad Abdallah Topic 13:Recombinant DNA Technology.
DNA Technology Chapter 20.
How do you identify and clone a gene of interest? Shotgun approach? Is there a better way?
15.2 Recombinant DNA Or How to Mess with DNA for Fun and Profit.
Technological Solutions. In 1977 Sanger et al. were able to work out the complete nucleotide sequence in a virus – (Phage 0X174) This breakthrough allowed.
Manipulating DNA.
Biotechnology Methods Producing Recombinant DNAProducing Recombinant DNA Locating Specific GenesLocating Specific Genes Studying DNA SequencesStudying.
Recombinant Technololgy
Remember the limitations? –You must know the sequence of the primer sites to use PCR –How do you go about sequencing regions of a genome about which you.
Genetic Engineering. What is genetic engineering? Application of molecular genetics for practical purposes Used to – identify genes for specific traits.
NIS - BIOLOGY Lecture 57 – Lecture 58 DNA Technology Ozgur Unal 1.
Cutting and Pasting DNA The cutters are called restriction enzymes, they cut DNA at specific nucleotide sequences.
Warm-Up #33 Answer questions #1-5 on Text page 321, Section Assessment.
DNA Technology. Overview DNA technology makes it possible to clone genes for basic research and commercial applications DNA technology is a powerful set.
19.1 Techniques of Molecular Genetics Have Revolutionized Biology
DNA TECHNOLOGY AND GENOMICS CHAPTER 20 P
Highlights of DNA Technology. Cloning technology has many applications: Many copies of the gene are made Protein products can be produced.
GENETIC ENGINEERING CHAPTER 20
Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment To work directly with specific genes, scientists prepare well-defined segments.
Chapter 10: Genetic Engineering- A Revolution in Molecular Biology.
Genetic Engineering Genetic engineering is also referred to as recombinant DNA technology – new combinations of genetic material are produced by artificially.
AP Biology Biotech Tools Review AP Biology Biotech Tools Review  Recombinant DNA / Cloning gene  restriction enzyme, plasmids,
Chapter 20: DNA Technology and Genomics - Lots of different techniques - Many used in combination with each other - Uses information from every chapter.
DNA Technology and Genomics
DNA Technology Ch. 20. The Human Genome The human genome has over 3 billion base pairs 97% does not code for proteins Called “Junk DNA” or “Noncoding.
DNA Technology & Genomics
Chapter 20 DNA Technology and Genomics. Biotechnology is the manipulation of organisms or their components to make useful products. Recombinant DNA is.
Gene Technologies and Human ApplicationsSection 3 Section 3: Gene Technologies in Detail Preview Bellringer Key Ideas Basic Tools for Genetic Manipulation.
RECOMBINANT DNA DNA THAT CONTAINS DNA SEGMENTS OR GENES FROM DIFFERENT SOURCES. DNA TRANSFERRED FROM ONE PART OF A DNA MOLECULE TO ANOTHER, FROM ONE CHROMOSOME.
Chapter 14 GENETIC TECHNOLOGY. A. Manipulation and Modification of DNA 1. Restriction Enzymes Recognize specific sequences of DNA (usually palindromes)
Lecture 3 – Selection of Recombinants & clone analysis The white colonies will all be recombinants, but only one of these many colonies will contain the.
- making changes to the DNA code of a living organism.
Chapter 13 Nucleic Acid Biotechnology Techniques Mary K. Campbell Shawn O. Farrell Paul D. Adams University.
15.2 Recombinant DNA. Copying DNA – How do scientists copy the DNA of living organisms? –The first step in using the polymerase chain reaction method.
Figure 20.0 DNA sequencers DNA Technology.
Chapter 7 Recombinant DNA Technology and Genomics
DNA Technologies (Introduction)
Section 3: Gene Technologies in Detail
COURSE OF MICROBIOLOGY
Ch. 13 Genetic Engineering
Chapter 20: DNA Technology and Genomics
DNA Tools & Biotechnology
Biotechnology CHAPTER 20.
DNA Technology Now it gets real…..
Gene Isolation and Manipulation
AMPLIFYING AND ANALYZING DNA.
Biotech Tools Review
Chapter 20 – DNA Technology and Genomics
Chapter 20 Biotechnology.
Chapter 14 Bioinformatics—the study of a genome
Recombinant DNA Technology
DNA Tools & Biotechnology
Recombinant DNA Technology
CHAPTER 20 DNA TECHNOLOGY.
Chapter 20: DNA Technology and Genomics
PowerLecture: Chapter 16
Presentation transcript:

DNA Technology- Cloning, Libraries, and PCR 14 and 16 November, 2005 Text Chapter 20

Cloning Overview DNA can be cloned into bacterial plasmids for research or commercial applications. The recombinant plasmids can be used as a source of DNA or, if a few rules are followed, can be used to express protein from any organism.

Restriction enzymes cut DNA at specific sequences Restriction enzymes cut DNA at specific sequences. Many restriction enzymes leave sticky ends - ends with single-stranded regions that are able to form base pairs with a complementary sequence.

Role of ampR and lacZ genes Cloning DNA into bacterial plasmids allows the bacteria to serve as factories, making large quantities of the plasmid of interest.

Start with a number of colonies, each carrying a plasmid with a different DNA fragment. A radioactive probe can be used to identify colonies that carry a plasmid that has an insert that is complementary to the probe. The single-stranded probe base pairs to any plasmid DNA that has complementary sequence. The fact that it is radioactive makes it easy to see where it went.

A cDNA contains only sequence that codes for protein.

DNA Libraries A library is a set of clones that carry different fragments, representing the entire genome of an organism (genomic library) or the mRNA expressed in a certain cell type at a certain time (cDNA library). Libraries can be constructed in plasmid or phage vectors.

Electrophoresis Agarose Gel Electrophoresis separates DNA fragments based on their size. DNA fragments are often detected using fluorescence.

Agarose gel electrophoresis can be used to investigate an individual’s genotype directly. If two alleles have sequence differences that change a restriction enzyme recognition site, then the size differences of the DNA fragments from a restriction digest can tell the researcher which alleles an individual carries. If this experiment is done on genomic DNA, then a radioactive probe complementary to this region is used to distinguish these fragments from the rest of the millions of fragments resulting from a digest of the genome.

The altered restriction site that produces the different sized fragments (an RFLP marker) does not have to be in the allele of interest. It simply has to be closely linked.

The Polymerase chain reaction can make a large number of copies of a specific sequence. The PCR reaction includes: Template DNA DNA Primers DNA Polymerase DNA monomers The PCR is often used to answer the same question that is answered by a radioactive probe - is a certain sequence present or not? If the sequence in question is present, a PCR product is made.

Southern Blotting

Determination of DNA sequence allows the researcher to determine genotype at the most fundamental level - the order of bases along the DNA molecule. This method uses DNA polymerase to synthesize new DNA strands in the presence of dideoxy nucleotides. Since these lack a 3’ OH group, whenever one is incorporated into the growing strand, that molecule does not elongate further.

Genome Sequencing Strategies

Early Conclusions from Genomics Assembly, annotation and prediction of genome sequence is computer-intensive. The pattern recognition and minimization algorithms are ideally suited to vector or SIMD hardware. Humans have far too few genes - about 30,000. Anternative splicing is important. The average gene is spliced in two or three different ways. Genetic similarity between organisms is striking. Predictions of relatedness based on morphology are sometimes upheld, challenged in other cases. Study of gene expression proceeds on a global level.

Microarray Hybridization

How was this experiment carried out? What are the conclusions?

Investigating the genotype of individuals can answer questions about phylogeny (relatedness). PCR Liquify mite Purify DNA Mt COI gene