Albia Dugger • Miami Dade College

Chapter 15Studying and

Manipulating Genes

15.1 Personal DNA Testing

• About 99% of your DNA is the same as everyone else’s

• A nucleotide difference found in at least 1% of a population is called a single-nucleotide polymorphism (SNP)

• SNPs account for many differences in the way humans look, and in the way our bodies work

Personal DNA Testing

• About 4.5 million SNPs in human DNA have been identified

• Using commercial SNP chips, individuals can now be tested for some of the SNPs they carry

• Personal genetic testing may soon allow physicians to customize treatments based on individual genetic makeup

Personal DNA Testing: SNP Chips

But how do you MAKE one of these?

CFU

START

15.2 Cloning DNA

• Researchers cut up DNA from different sources, then paste the resulting fragments together

• Cloning vectors can carry foreign DNA into host cells

Cutting and Pasting DNA

• Restriction enzymes• Bacterial enzymes that cut DNA

• Recombinant DNA• Composed of DNA from two or more organisms

Making Recombinant DNA

A restriction enzyme recognizes a specific base sequence (orange boxes) in DNA from any source.

1

restriction enzyme (cut)

The enzyme cuts DNA from two sources into fragments. This enzyme leaves sticky ends.

2

mix

When the DNA fragments from the two sources are mixed together, matching sticky ends base-pair with each other.

3

DNA ligase(paste)

DNA ligase joins the base-paired DNA fragments. Molecules of recombinant DNA are the result.

4

Stepped Art

DNA Cloning

• Making recombinant DNA is the first step in DNA cloning

• DNA cut is inserted into cloning vectors (plasmids) cut with the same enzyme

• Cloning vectors with foreign DNA are placed in host cells clones

Figure 15-3 p236

pDrive Cloning Vector3.85 kb

Kpn l

Sph l

Pst l

Bam Hl

Eco RI

Sal l

Acc l

Xho l

Xba l

Bst XI

Sac l

Not l

lac

Figure 15-3a p236

DNA Cloning

recombinant plasmid

plasmid cloning vector

chromosomal DNA

A A restriction enzyme cuts a specific base sequence in chromosomal DNA and in a plasmid cloning vector.

cutplasmid

chromosomalDNA fragments

B A fragment of chromosomal DNA and the plasmid base-pair at their sticky ends. DNA ligase joins the two pieces of DNA.

Stepped Art

C The recombinant plasmid is inserted into a host cell. When the cell multiplies, it makes multiple copies of the plasmids.

+

cDNA Cloning

EcoRI recognition site

cDNA

mRNA

cDNA

mRNA

DNA

15.3 Isolating Genes

• DNA libraries and the polymerase chain reaction (PCR) help researchers isolate particular DNA fragments

DNA Libraries

• Genome• The entire set of genetic material of an organism

• DNA libraries are sets of cells containing various cloned DNA fragments• Genomic libraries (all DNA in a genome)• cDNA libraries (all active genes in a cell)

Probes

• Probe• A fragment of DNA labeled with a tracer• Used to find a specific clone carrying DNA of interest in a

library of many clones

• Nucleic acid hybridization• Base pairing between DNA from different sources• A probe hybridizes with the targeted gene

Figure 15-5 p238

D A probe is added to the liquid bathing the paper. The probe hybridizes (base-pairs) with the spots of DNA that contain complementary base sequences.

C The paper is soaked in a solution that ruptures the cells and releases their DNA. The DNA clings to the paper in spots mirroring the distribution of colonies.

B A piece of special paper pressed onto the surface of the growth medium will bind some cells from each colony.

A Individual bacterial cells from a DNA library are spread over the surface of a solid growth medium. The cells divide repeatedly and form colonies—clusters of millions of genetically identical descendant cells.

E The bound probe makes a spot. Here, one radioactive spot darkens x-ray film. The position of the spot is compared to the positions of the original bacterial colonies. Cells from the colony that made the spot are cultured, and the DNA they contain is harvested.

PCR

• Polymerase chain reaction (PCR)• A cycled reaction that uses a heat-tolerant form of DNA

polymerase (Taq polymerase) to produce billions of copies of a DNA fragment

PCR Techniques

• DNA to be copied is mixed with DNA polymerase, nucleotides and primers that base-pair with certain DNA sequences

• Cycles of high and low temperatures break and reform hydrogen bonds between DNA strands, doubling the amount of DNA in each cycle

Two Rounds of PCR

15.4 DNA Sequencing

• DNA sequencing reveals the order of nucleotide bases in a fragment of DNA

DNA Sequencing

• DNA is synthesized with normal nucleotides and dideoxynucleotides tagged with different colors

• When a tagged base is added, DNA synthesis stops; fragments of all lengths are made

• Electrophoresis separates the fragments of DNA, each ending with a tagged base, by length

• The order of colored bases is the sequence of DNA

Figure 15-7a p240

DNA template

2

3

4

5

Figure 15-7 p240

dideoxynucleotides1

base:

pigment:

adenine

green

cytosine

blue

guanine

black

thymine

red

The Human Genome Project

• Automated DNA sequencing and PCR allowed human genome projects to sequence the 3 billion bases in the human genome

• 28,976 genes have been identified, but not all of their products or functions are known

Sequencing the Human Genome

A Human DNA Sequence

15.5 Genomics

• Comparing the sequence of our genome with that of other species is giving us insights into how the human body works

• Unique sequences of genomic DNA can be used to distinguish an individual from all others

Genomics

• The study of genomes (genomics) is a broad field that encompasses whole-genome comparisons, structural analysis of gene products, and surveys of small-scale variations in sequence

Comparing Genomes

• All genomes are related to some extent – comparing genomes provides evidence of genetic relationships

• Comparing genomes among species also shows that changes in chromosome structure do not occur randomly

• Comparing the coding regions of genomes also offers medical benefits• Example: APOA5 mutations and triglycerides

Genomic DNA Alignment

DNA Profiling: SNPs

• Identifying an individual by his or her unique array of DNA sequences is called DNA profiling

• One type of DNA profiling involves SNP-chips with microscopic spots of DNA stamped on them

• An individual’s genomic DNA hybridizes only with DNA spots that have a matching SNP sequence

• Probes reveal where the genomic DNA has hybridized

SNP-Chip Analysis

DNA Profiling: STRs

• Another method of DNA profiling involves analysis of short tandem repeats, sections of DNA in which a series of 4 or 5 nucleotides is repeated several times in a row.

• Types and numbers of STRs vary greatly among individuals

• Unless two people are identical twins, the chance that they have identical short tandem repeats in even three regions of DNA is 1 in a quintillion (1018)

Analyzing STRs

• PCR is used to amplify DNA from regions of several chromosomes that have STRs

• Electrophoresis is used to separate the fragments and create a unique DNA fingerprint

• DNA fingerprints have many applications, including legal cases, forensics, and population studies

An STR Profile

15.6 Genetic Engineering

• Genetic engineering is a laboratory process by which deliberate changes are introduced into an individual’s genome

Some GMOs

• The most common GMOs are bacteria and yeast

• Some E. coli have been modified to produce a fluorescent protein from jellyfish – used to study gene expression

• Some bacteria have been modified to produce medically important proteins such as human insulin and chymotrypsin

• Other GMO-produced enzymes improve the taste and clarity of beer and fruit juice, slow bread staling, or modify fats

E. coli with Jellyfish Genes

15.7 Designer Plants

• Genetically engineered crop plants are widespread in the United States

Introducing New Genes into Plants

• Foreign or modified genes can be introduced into plant cells by way of electric or chemical shocks, or by blasting them with DNA-coated micropellets

• Genes can also be introduced biologically using the Ti plasmid, a plasmid of the bacterium Agrobacterium tumefaciens which contains tumor-inducing (Ti) genes

Figure 15-12a p245

A A Ti plasmid carrying a foreign gene is inserted into an Agrobacterium tumefaciens bacterium.

Figure 15-12b p245

B The bacterium infects a plant cell and transfers the Ti plasmid into it. The plasmid DNA becomes integrated into one of the cell’s chromosomes.

Figure 15-12c p245

C The plant cell divides, and its descendants form an embryo. Several embryos are sprouting from this mass of cells.

Figure 15-12d p245

D Each embryo develops into a transgenic plant that expresses the foreign gene. The glowing tobacco plant is expressing a gene from fireflies.

Genetically Engineered Plants

• Crop plants are genetically modified to produce more food at lower cost• Resistance to disease or herbicides• Increased yield• Plants that make pesticides (Bt protein)• Drought resistance

• Genetic modifications can make food plants more nutritious• Rice plants that make β-carotene

GMO Controversies

• Many people are opposed to any GMO

• The USDA Animal and Plant Health Inspection Service (APHIS) has approved 78 GMO crop plants for use in US, including corn, sorghum, cotton, soy, canola, alfalfa

• In crops engineered for herbicide resistance, weeds are becoming resistant to herbicides

• Pollen containing engineered genes is spreading into wild plants and nonengineered crops

Corn with the Bt Gene

15.8 Biotech Barnyards

• Genetically engineered animals are invaluable in medical research and in other applications

Transgenic Animals

• Animals that would be impossible to produce by traditional breeding methods are being created by genetic engineering

• The first genetically modified animals were mice used in medical research as models of human diseases

• Genetically modified animals also make proteins with medical and industrial applications

Examples of Transgenic Animals

• Transgenic mice are used in knockout experiments to reveal the functions of human genes

• Transgenic goats produce proteins used to treat cystic fibrosis, heart attacks, and blood clotting disorders

• Milk from transgenic goats contains lysozyme or spider silk

• Transgenic rabbits make human interleukin-2

• Transgenic livestock have many altered qualities

Some Genetically Modified Animals

Brain of Mouse Transgenic for Multiple Pigments

Knockouts and Organ Factories

• Transgenic pigs with human proteins that are not rejected by the human immune system are a potential source of organs and tissues for transplants in humans

• Transplantation of a tissue or organ from one species to another is called xenotransplantation

• Critics are concerned that pig-to-human transplants would allow pig viruses to cross the species barrier and infect humans, perhaps with catastrophic results

15.9 Safety Issues

• The first transfer of foreign DNA into bacteria ignited an ongoing debate about potential dangers of transgenic organisms that may enter the environment

Into the Unknown

• Early questions about new technologies:• Could genetic recombination in laboratories produce a

new superpathogen?• What if new forms escaped into the environment and

transformed other organisms?

• Safety guidelines for genetic engineering were adopted in the US and other countries

15.10 Genetically Modified Humans

• We as a society continue to work our way through the ethical implications of applying new DNA technologies

• The manipulation of individual genomes continues even as we are weighing the risks and benefits of this research

Gene Therapy

• Gene therapy is now being tested as a treatment for several types of cancer, sickle-cell anemia, cystic fibrosis, and other inherited diseases

• Gene therapy• Transfer of recombinant DNA into body cells to correct a

genetic defect or treat a disease• Viral vectors or lipid clusters insert an unmutated gene into

an individual’s chromosomes

Getting Better

• Children with severe combined immunodeficiency disease (SCID-X1) can survive only in germ-free isolation tents

• 1998: A viral vector was used to insert unmutated IL2RG genes into 20 boys with SCID-X1

• Within months 18 boys left their isolation tents for good – gene therapy permanently repaired their immune systems

Immune System Repaired by Gene Therapy

Getting Worse

• No one can predict where a virus-injected gene will insert into a chromosome

• Five of the 20 boys treated with gene therapy for SCID-X1 developed cancer (leukemia), and one of them has died

• In other studies, severe allergic reactions to the viral vector itself have resulted in death

Getting Perfect

• Eugenics – the selection of human being with the most desirable traits – has been used as a justification for horrific acts such as genocide

• It may soon be possible to engineer humans for particular desirable traits such as height, strength, or intelligence

• Many questions must be answered about the ethics and consequences of manipulating the human genome

Getting Perfect

• Would it be okay to genetically engineer “superhumans”?