Chapter 5 bio 300 obe

Chapter 5 : DNA TECHNOLOGY1

Chapter 5 : DNA TECHNOLOGY 2

BIO 300BIO 300BIOLOGICAL TECHNIQUES AND SKILLSBIOLOGICAL TECHNIQUES AND SKILLS

SARINI BINTI AHMAD WAKIDSARINI BINTI AHMAD WAKIDFACULTY OF APPLIED SCIENCEFACULTY OF APPLIED SCIENCE


CHAPTER 5CHAPTER 5TECHNIQUES in DNA TECHNOLOGYTECHNIQUES in DNA TECHNOLOGY


DNA SEQUENCING The ultimate level of analysis is determination

of the actual sequence of bases in a DNA molecule. The development of sequencing has paralleled the advancement of molecular biology. The genomics was born out of the ability to determine the sequence of an entire genome relatively rapid.


Idea To generate a set of nested fragments that

each begin with the same sequence and end in a specific base. When this set of fragments is separated by high resolution gel electrophoresis, the results is a “ladder” of fragments in which each band consist of fragments that end in a specific base. By starting with the shortest fragments, one can read the sequence by moving up the ladder.


ENZYMATIC SEQUENCING

Developed by Fredrick Sanger, who also was the first to determine the complete sequence of a protein.

This method uses dideoxynucleotides as chain terminators in DNA synthesis reactions. A dideoxynucleotides has H in place of OH at both the 2’ position and at the 3’ position.


AUTOMATED SEQUENCING

The technique of enzymatic sequencing is very powerful, but it is also labor intensive and takes a significant amount of time. It requires a series of enzymatic manipulations, time for electrophoresis, then time to expose the gel to film.


PCRPCRPolymerase Chain ReactionPolymerase Chain Reaction


Review: The structure of DNA

Helix Complementary Base Pairing


Review: The structure of DNA

Antiparallel Strands

Unzipping


Review: Genome Sizes

Pine: 68 billion bp Corn: 5.0 billion bp Soybean: 1.1 billion bp Human: 3.4 billion bp Housefly: 900 million bp Rice: 400 million bp E. coli: 4.6 million bp HIV: 9.7 thousand bp

http://www.cbs.dtu.dk/databases/DOGS/abbr_table.txt


Just How Big Is 3.4 Billion?

Human genome is 3.4 B bp If the bases were written in standard

10-point type, on a tape measure... ...The tape would stretch for 5,366

MILES!

Identifying a 500bp sequence in a genome would be like finding a section of this tape measure only 4 feet long!


The Problem...

How do we identify and detect a specific sequence in a genome?

TWO BIG ISSUES: There are a LOT of other sequences in a genome

that we’re not interested in detecting. The amount of DNA in samples we’re interested in is

VERY small.

PCR solves BOTH of these issues!!!

SPECIFICITY

AMPLIFICATION


PCRPCR

- a method for amplifying (copying) small - a method for amplifying (copying) small amount of DNA in nearly any amount amount of DNA in nearly any amount required, starting with a small initial quantity.required, starting with a small initial quantity.

- an in vitro or cell-free method for - an in vitro or cell-free method for synthesizing DNA.synthesizing DNA.

- it was invented in 1985 by Kary Mullis - it was invented in 1985 by Kary Mullis (received the Nobel Prize for chemistry in (received the Nobel Prize for chemistry in 1993).1993).


PCR History

In what has been called by some the greatest achievement of modern molecular biology, Kary B. Mullis developed the polymerase chain reaction (PCR) in 1983. PCR allows the rapid synthesis of designated fragments of DNA. Using the technique, over one billion copies can be synthesized in a matter of hours.

PCR is valuable to scientists by assisting gene mapping, the study of gene functions, cell identification, and to forensic scientists in criminal identification. Cetus Corporation, Mullis' employer at the time of his discovery, was the first to commercialize the PCR process. In 1991, Cetus sold the PCR patent to Hoffman-La Roche for a price of $300 million. It is currently an indispensable tool for molecular biologists and the development of genetic engineering.


Idea Simple Two primers are used that complimentary to the

opposite strands of a DNA sequence, oriented toward each other. When DNA polymerase acts on these primers and the sequence of interest, the primers produce complementary strands, each containing the other primer. If this procedure is done cyclically, the result is a large quantity of a sequence corresponding to the DNA that lies between the two primers.



PCR Machine / ThermocyclerPCR Machine / Thermocycler


PCRPCR

Components of PCRComponents of PCR Template DNA primers dNTPs (dATP, dTTP, dCTP & dGTP) Taq DNA polymerase MgCl2

PCR buffer, pH 8


PCRPCR

Three major phases in PCR: Three major phases in PCR: Denaturing –high temperature (94ºC) Annealing of primers –low temperature(55ºC) Extension – synthesis – intermediate

temperature (72ºC)

The total time to perform a standard PCR is The total time to perform a standard PCR is approximately 4 hours. approximately 4 hours.




Factors influencing PCRFactors influencing PCR

Quality of template DNA Concentration of template DNA Primers Concentration of MgCl2

Annealing temperature


Quality of template DNAQuality of template DNA

- should be free of proteases that could degrade the DNA polymerase.

- template DNA with high levels of proteins or salts should be diluted or cleaned up to reduce inhibition of DNA polymerase activity.


Concentration of template DNAConcentration of template DNA

- highly concentrated template DNA may yield nonspecific product or inhibit the reaction.

- it is rare that template DNA concentration is too low.


PrimersPrimers

- select primers with a random base distribution and GC content similar to template DNA being amplified.

- avoid sequences with secondary structure, especially at the 3’ end.

- check primers for complementary and avoid primers with 3’ overlaps to reduce primer-dimer artifacts.

- design so the base at the 3’ end of the primer is a G or C to enhance specificity.


Concentration of MgClConcentration of MgCl22

- MgCl2 concentration is very important.

- excess Mg2+ promotes production of nonspecific product and primer-dimer artifacts.

- insufficient Mg2+ reduces yield.


Annealing temperatureAnnealing temperature

- annealing temperature depends on length and GC content of primers (55ºC good for primers 20 nucleotides long; 50%).

- Higher annealing temperatures may be needed to increase primer specificity.


Some Uses of PCR

Forensic DNA detection Identifying transgenic plants Detection and quantification of

viral infection Cloning Detection of ancient DNA Gene expression analysis


Uses of PCR: Ancient DNA

http://www.faseb.org/opar/bloodsupply/pcr.html

Archaeologists have happily seized on PCR and are applying it in an amazing variety of ways. It is helping, for example, to launch a new chapter in the colorful and controversial story of the 2000-year-old Dead Sea Scrolls, which are written on parchment made out of skins from goats and gazelles. Researchers are analyzing the parchment fragments to try to identify individual animals they came from. The hope is that the genetic information will guide them in piecing together the 10,000 particles of scrolls that remain.


Uses of PCR: Disease Detection


PCR can also be more accurate than standard tests. It is making a difference, for example, in a painful, serious, and often stubborn misfortune of childhood, the middle ear infection known as otitis media. The technique has detected bacterial DNA in children's middle ear fluid, signaling an active infection even when culture methods failed to detect it. Lyme disease, the painful joint inflammation caused by bacteria transmitted through tick bites, is usually diagnosed on the basis of symptom patterns. But PCR can zero in on the disease organism's DNA contained in joint fluid, permitting speedy treatment that can prevent serious complications.


Uses of PCR: Endangered Species


Researchers have used the technique to aid in reducing illegal trade in endangered species, and products made from them. Because PCR is a relatively low-cost and portable technology, and likely to become more so, it is adaptable for field studies of all kinds in the developing countries. It is also a tool for monitoring the release of genetically engineered organisms into the environment.


Uses of PCR: Forensic DNA


The technique's unparallelled ability to identify and copy the tiniest amounts of even old and damaged DNA has proved exceptionally valuable in the law, especially the criminal law. PCR is an indispensable adjunct to forensic DNA typing-commonly called DNA fingerprinting.


Uses of PCR: Proving Innocence


DNA typing is only one of many pieces of evidence that can lead to a conviction, but it has proved invaluable in demonstrating innocence. Dozens of such cases have involved people who have spent years in jail for crimes they did not commit. Many people have been freed because of the power of PCR. Even when evidence such as semen and blood stains is years old, PCR can make unlimited copies of the tiny amounts of DNA remaining in the stains for typing.




The method is especially useful for searching out disease organisms that are difficult or impossible to culture, such as many kinds of bacteria, fungi, and viruses, because it can generate analyzable quantities of the organism's genetic material for identification. It can, for example, detect the AIDS virus sooner during the first few weeks after infection than the standard ELISA test. PCR looks directly for the virus's unique DNA, instead of the method employed by the standard test, which looks for indirect evidence that the virus is present by searching for antibodies the body has made against it.


Uses of PCR: Ancient DNA


Archaeologists are finding that PCR can illuminate human cultural practices as well as human biology. Analyzing pigments from 4000-year-old rock paintings in Texas, they found one of the components to be DNA, probably from bison. The animals did not live near the Pecos River at that time, so the paleo-artists must have gone to some effort to obtain such an unusual ingredient for their paint. Taking so much trouble suggests that the paintings were not simply decorations, but had religious or magical significance.




PCR can even diagnose the diseases of the past. Former vice president and presidential candidate Hubert H. Humphrey underwent tests for bladder cancer in 1967. Although the tests were negative, he died of the disease in 1978. In 1994, researchers compared a 1976 tissue sample from his cancer-ridden bladder with his 1967 urine sample. With the help of PCR amplification of the small amount of DNA in the 27-year-old urine, they found identical mutations in the p53 gene, well-known for suppressing tumors, in both samples. "Humphrey's examination in 1967 may have revealed the cancerous growth if the techniques of molecular biology were as well understood then as they have become," the researchers said.


Uses of PCR: Gene Expression Analysis

The Human Genome Project has identified tens of thousands of genes in the human genome. A key questions is: what do these genes do? Part of the answer comes from determining when the genes are turned on and off, and what affects the level of gene expression. Quantitative PCR is a key component of determining the levels of gene expression, and is a critical tool in cancer research, disease studies, and developmental biology.

DNARNA

Enzymes

GENEX AnalysisBiology


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Chapter 5 bio 300 obe

Technology