Biogenetic Engineering & Manipulating Genes

Chapter 20

Intro. Q’s #6-for Chapter 20: Genetic Engineering1) What does the acronym PCR stand for and what does this process

do?2) What does Gel electrophoresis allow us to do?3) Give two applications of DNA profiling.4) What are the advantages and disadvantages of genetic screening?5) Describe what genetic engineering is and explain how such items as

restriction enzymes, DNA ligase, and the production of “sticky ends” are used.

6) Name two “vectors” that can be used for gene transfer.7) Give two examples of genetically modified crops or animals8) Briefly explain the process of gene therapy and give an example

how it works.9) Explain what a clone is and how it could be formed.10) What are some of the ethical concerns about cloning? Give your

opinion if you think cloning is something we should be doing.

Genetic Engineering

• Chapter 14• DNA Technology &

Genomics

O.J. Simpson capital murder case,1/95-9/95• Odds of blood in Ford Bronco not being R. Goldman’s:

• 6.5 billion to 1• Odds of blood on socks in bedroom not being N. Brown-Simpson’s:

• 8.5 billion to 1• Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:

• 21.5 billion to 1• Number of people on planet earth:

• 6.1 billion• Odds of being struck by lightning in the U.S.:

• 2.8 million to 1• Odds of winning the Illinois Big Game lottery:

• 76 million to 1 • Odds of getting killed driving to the gas station to buy a lottery ticket

• 4.5 million to 1• Odds of seeing 3 albino deer at the same time:

• 85 million to 1• Odds of having quintuplets:

• 85 million to 1• Odds of being struck by a meteorite:

• 10 trillion to 1

Recombinant DNA

• Def: DNA in which genes from 2 different sources are linked

• Genetic engineering: direct manipulation of genes for practical purposes

• Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Restriction Enzymes

Tools of Genetic Engineering• Restriction enzymes (endonucleases)

-in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific

• Restriction site:-recognition sequence for a particular restriction enzyme

• Restriction fragments:-segments of DNA cut by restriction enzymes in a reproducible way

• Sticky end:-short extensions of restriction fragments

• DNA ligase: -enzyme that can join the sticky ends of DNA fragments

• Cloning vector: -DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

Producing Restriction Fragments• DNA ligase used to splice together cut plasmids

and chromosome fragments

• After amplification, clones are:

-identified

-some are always stored in a genomic library

Tools for DNA Analysis & Genomics

• PCR (polymerase chain reaction)

• Gel electrophoresis

• Restriction fragment analysis (RFLPs)

• Southern blotting

• DNA sequencing

• Human genome project

Polymerase Chain Reaction (PCR)http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

• Amplification of any piece of DNA without cells (in vitro)

• Produces many identical copies of a DNA segment

• Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers

• Applications: fossils, forensics, prenatal diagnosis, etc.

Polymerase Chain reaction (PCR)

Polymerase Chain Reaction

• PCR = common method of creating copies of specific fragments of DNA– PCR rapidly amplifies a single DNA molecule into

many billions of molecules.

• Small samples of DNA can produce sufficient copies to carry out forensic tests

DNA Profiling (DNA fingerprinting)

Two Applications:

-Used in criminal investigations

-Identify the remains of dead people

Restriction Fragment Analysis• Restriction fragment length polymorphisms (RFLPs)

• Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence

• DNA Fingerprinting (DNA Profiling)

Gel Electrophoresis

• DNA fragments placed into “wells” in gel agarose• Electricity pulls on DNA fragments• Fragments travel at

different rates based on size and ability to squeeze through swiss-cheese-like agarose

Gel Electrophoresis• separates nucleic acids or proteins on the basis of size and electrical charge

creating DNA bands of the same length

• DNA has a net negative charge (use a positive charge in the gel)

Applications of RFLPs

• DNA cut by restriction enzymes & separated on gel electrophoresis

• Distinct banding patterns reveal the slight variations of DNA

• Makes each individual identifiable

Applications of RFLPs

• Restriction Fragment Length Polymorphism

• RFLPs have increased sites available for mapping the human genome

Applications of RFLPs

• RFLP analysis identifies mutant alleles• RFLP analysis reveals a unique genetic fingerprint

useful in solving cases of parenthood, rape, and murder

DNA Sequencing

• Determination of nucleotide sequences (Sanger method, sequencing machine)

• Genomics: the study of genomes based on DNA sequences

• Human Genome Project

Practical DNA Technology Uses• Diagnosis of disease• Human gene therapy• Pharmaceutical products

(vaccines)• Forensics• Animal husbandry

(transgenic organisms)• Genetic engineering in plants• Ethical concerns?

Genetic ScreeningDef: Testing individuals in a population for the presence or

absence of a gene (allele)Advantages:

-pre-natal diagnosis of genetic disorders-Could help stop the spread of a disorder-Can detect carriers of a potential disorder

Disadvantages:-invasion of privacy-Individuals can become stigmatized in the community-Discriminated against or feared-Employment and medical insurance

Cloning

Bacterial plasmids in gene cloning

Steps for Eukaryotic gene cloning

Plant Cloning

• Tissue Culture Propagation– Bits of phloem can

be induced in the lab to form clumps of tissue that will make roots & shoots

– Orchid culture

Embryo Cloning

• Medical technique which produces identical twins or triplets– Duplicates nature

– One or more cells are removed from a fertilized embryo, encouraged to develop into one, identical twins or triplets

– Done for many years on animals

– Limited experimentation on humans

Adult DNA Cloning

• Untried on humans-potential of producing a twin of an existing person

Therapeutic Cloning

• Stem cells removed from an embryo with intent of producing tissue or a whole organ for transplant back into the person who supplied the new DNA– Embryo dies in the process

• Goal is to produce a healthy copy of a sick person's tissue or organ for transplant

Therapeutic Cloning

• Vastly superior to organ transplants– Supply would be unlimited - no waiting lists

• Tissue or organ would have the sick person's original DNA– No immunosuppressant

drugs would need to be taken

Adult DNA Cloning

Adult DNA Cloning

Stem

cell

clon-ing

Theraputic Stem Cell Cloning

• Used

Intro. Q’s for Chapter 14: Genetic Engineering1) What does the acronym PCR stand for and what does this process

do?2) What does Gel electrophoresis allow us to do?3) Give two applications of DNA profiling.4) What are the advantages and disadvantages of genetic screening?5) Describe what genetic engineering is and explain how such items as

restriction enzymes, DNA ligase, and the production of “sticky ends” are used.

6) Name two “vectors” that can be used for gene transfer.7) Give two examples of genetically modified crops or animals8) Briefly explain the process of gene therapy and give an example

how it works.9) Explain what a clone is and how it could be formed.10) What are some of the ethical concerns about cloning? Give your

opinion if you think cloning is something we should be doing.

Chromatin

• Def: complex of DNA and proteins• DNA Packing• Histone proteins (+ charged amino acids w/ phosphates of DNA

that are - charged)• Nucleosome

-”beads on a string”; basic unit of DNA packing

• Heterochromatin -highly condensed interphase DNA

(can not be transcribed)• Euchromatin

-less compacted interphase DNA (can be transcribed)

DNA Libraries• Collection of DNA fragments that have been

incorporated into plasmids

Steps for Eukaryotic Gene Cloning• Isolation of cloning vector (bacterial plasmid) &

gene-source DNA (gene of interest)

• Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase

• Introduction of cloning vector into cells (transformation by bacterial cells)

• Cloning of cells (and foreign genes)

• Identification of cell clones carrying the gene of interest