DNA TECHNOLOGYCHAPTER 20

CampbellChapter 13 Mader

What is it?

• DNA technology is the chemical manipulation of the genotypes and resulting phenotypes of organisms such that living organisms are modified; alternatively, no-longer-living organisms or their no-longer-living parts may be analyzed chemically at the level of genotype

Why important?

• The use of DNA technology has revolutionized how scientists study the genetics, biochemistry, even the ecology and evolutionary biology of organisms, plus has allowed the development of novel biological products, indeed whole industries are now devoted to DNA-technology-based production and analysis of biological materials

Genetic Engineering is…

• the artificial manipulation of the genetic material of organisms, including the creation of novel genetic material (i.e., novel nucleotide sequences)

• This manipulation occurs to a large extent external to organisms, e.g., in test tubes, a.k.a., in vitro (meaning, literally, “in glass”)

Genetic Engineering is used to…

• Make recombinant DNA

• To purposefully change nucleotide sequences

• To clone DNA

-manipulation of an organism’s genotype by artificial, typically very direct means

Biotechnology is …

– Contrasting with genetic engineering, biotechnology refers to the engineering of phenotype

– Biotechnology is not limited to the manipulation of phenotype by directly manipulating genotype (i.e., via genetic engineering) though today this is very often how phenotypes are manipulated (often to the detriment of more traditional means such as plant and animal breeding)

Molecular techniques – there are many!

• Gene cloning - rDNA (and is associated with various molecular techniques—including in vitro restriction enzyme digests, and DNA ligation—plus additional, less-artificial manipulations including transformation and transduction)

• Creation of cDNA• Polymerase chain reaction• Gel electrophoresis• Various blotting techniques (Southern blotting,

Northern blotting, Western blotting, etc.) • RFLP analysis• DNA sequencing

Isolating DNA

• fairly straightforward involving the breaking open of cells and subsequent purification of the DNA component

• The actual molecular manipulation of DNA begins only once the DNA is purified, and involves to a large extent the cutting of DNA at specific nucleotide sequences by proteins known as restriction enzymes

Restriction Enzymes and the Restriction Fragment

• The actual nucleotide sequence on a piece of DNA that a restriction enzyme cuts is called a restriction site

• Most restriction sites are palindromes with identical sequences regardless of the direction one moves down the DNA (keeping in mind, of course, that DNA is antiparallel such that one moves down or up a different strand if one switches direction : What are some palindromes in English language?

How the RE cuts

• RE are used (and harvested) for their specific cutting ability

• In order to make rDNA – need nice sticky end but blunt ends are produced too

Restriction fragment length polymorphism (RFLP)

• The location of restriction sites in a genome occurs fairly randomly, and can differ from person to person

• differences in nucleotide sequences at specific loci found within populations polymorphisms

RFLPs Importance

• As a consequence of these differences between individuals in the location of specific restriction sites, the distance between sites will vary, thus length of restriction fragments produced by digesting an individual’s genome using specific restriction enzymes will also vary

• The variation between individuals is called restriction fragment length polymorphism

Remember OJ – the glove!

• Since the nucleotide sequence of nearly every individual is unique, the RFLPs of each individual are also unique, and thus RFLP analysis may be employed to forensically distinguish individuals (hence the synonymous term, DNA fingerprinting)

Comparing the band patterns!

Gel Electrophoresis

• Gel electrophoresis is a method of molecule (or complex) separation in which molecules move within a gel-like medium (think jello or, even better, the agar found in a petri dish)

Gel Electrophoresis Continued

– In gel electrophoresis charged molecules are pulled through the gel (typically consisting of the polymer polyacrylamide or of purified agar known as agarose)

• Larger or more diffuse molecules move more slowly because they tend to get hung up in the gel matrix

• Molecules with greater charges also move faster because it is an electric voltage that is employed to pull the molecules through the gel matrix

OVERVIEW

• http://web.utk.edu/~khughes/GEL/sld001.htm

Gene Cloning –the steps

• Isolating DNA from the cell of an organism (including digestion with restriction enzymes)

• Insertion of that DNA into a plasmid• Placement of the plasmid into a second cell• Measures taken to make sure that the cloned

DNA is the DNA of interest• Various manipulations of the DNA including

subcloning, sequencing, and expression

How to make rDNA – remember we need those sticky ends

• http://www.biology87.org/apbio/molgen/Activity6_notes.pdf

Many purposes for rDNA

• Figure 20.1– Protein harvesting– Alter genetic make-up– Resistance– Cloning a gene of interest (fig. 20-3)

PCR= Polymerase Chain Reaction

• In polymerase chain reaction, one employs short DNA primers that are complementary to the opposite ends of a specific sequence of DNA one in interested in amplifying in number (again, keep in mind that DNA is antiparallel and that consequently opposite ends means also complementary to opposite strands)

PCR continued

• The primers supply the 3’ –OH “primer” necessary for the initiation of DNA replication

• Polymerization is used to produce double-stranded DNA (i.e., a double helix) using single-stranded DNA as the template

Still PCR

– Individual single-strands of DNA are typically formed via the unwinding of a DNA double helix by the application of heat

– Thus, PCR consists of heat treatment to unwind DNA, binding of primers to the resulting single-stranded DNA, polymerization of new DNA to form a new double-stranded DNA double helix, repeat

See Methods: The Polymerase Chain Reaction

(PCR) found on page 371

• Neat things about PCR

• Can be performed in a single reaction vessel to which one has to add the various necessary ingredients only once—thereby repeated rounds of synthesis may be effected simply by heating and cooling the reaction vessel

Another neat thing about PCR

• may be initiated using only very small, impure samples of DNA

-as in dinosaur DNA, or teeny tiny samples from a crime scene (even partial pieces)

Southern Blotting

• Combo of methods– DNA isolation and _________________________– DNA run using ____________________________– _____________ - use alkaline solution and sponge to

pull DNA fragments onto nitrocellulose paper, denatures the DNA into single strands

– _________________________________ –radioactive single stranded DNA that is complementary to the gene of interest – a solution of these probes

– ____________________________ - photo image of the radioactive bands ONLY!

WHY USED?

• http://www.dnalc.org/ddnalc/resources/shockwave/southan.html

Watch animation and summarize how it is used in forensics:

SANGER METHOD

• Used to determine___________________

• How it works:– One piece of DNA is divided into ___ portions– Each portion is placed in a mixture that

contains all enzymes and primers needed to synthesize cDNA

– Key difference between TT, __________________________________

Sanger Continued

– Each TT contains the same DNA but• TT1 – radioactive ___’s • TT2 – radioactive ___’s• TT3 – radioactive ___’s• TT4 – radioactive ___’s

- The marked DNA are run using highly specific agarose that separates the DNA into __________________________.

Reading the nucleotide sequence

• http://www.zerobio.com/sanger1.htm

You try it!

• What is the DNA sequence?

Practical Applications – present Tuesday

• Medical Contributions– Diagnosis of disease– Human Gene Therapy– Pharmaceuticals

• Forensics, Environmental, Agriculture– CSI– Environmental– Agriculture (3 people)

• Safety and ethical issues

TEST on Chapter 20, parts of 18 and 19 on Weds.

• Mon – finish biotech techniques

• Tues – quick presentations on Apps.

• Weds – Test on 18, 19 and 20!

• Thursday – Bioterrorism Holiday Party!» Half-year review guide» Essay practices – three will be selected for the final

exam. Write outline for all of them.