www.cengage.com/biology/starr

Albia Dugger • Miami Dade College

Cecie StarrChristine EversLisa Starr

Chapter 15Biotechnology

(Sections 15.1 - 15.5)

15.1 Personal DNA Testing

• Personal DNA testing companies identify a person’s unique array of single-nucleotide polymorphisms (SNPs) – some of which are related to risk of diseases such as Alzheimer’s

• Personal genetic testing may soon revolutionize medicine by allowing physicians to customize treatments on the basis of an individual’s genetic makeup

Personal DNA Testing

• This chip reveals which versions of 906,600 SNPs occur in the individual’s DNA

ABC Video: DNA Mystery: Human Chimeras

ABC Video: Family Ties: Paternity Testing

15.2 Cloning DNA

• Researchers use restriction enzymes to cut up DNA, then bond the fragments together using DNA ligase

• Fragments with complementary tails (“sticky ends”) stick together when their matching tails base-pair

• restriction enzyme • Bacterial enzyme used to cut specific nucleotide

sequences in DNA

Recombinant DNA

• DNA fragments from different organisms combine to make a hybrid molecule: recombinant DNA

• recombinant DNA • A DNA molecule that contains genetic material from more

than one organism

Making Recombinant DNA (1)

• A restriction enzyme recognizes a specific base sequence in DNA from any source

Making Recombinant DNA (2)

• The enzyme cuts DNA from two sources into fragments

• The enzyme leaves sticky ends

Making Recombinant DNA (3)

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

Making Recombinant DNA (4)

• DNA ligase joins the base-paired DNA fragments

• Molecules of recombinant DNA result

Fig. 15.2, p. 220

restriction enzyme (cut)

mix DNA ligase(paste)

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

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

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

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

1 2 3 4

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

Fig. 15.2, p. 220

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

Making Recombinant DNA

Animation: Restriction Enzymes

DNA Cloning

• DNA cloning mass produces specific DNA fragments

• Fragments to be copied are inserted into plasmids or other cloning vectors and inserted into host cells such as bacteria

• Host cells divide and make identical copies (clones) of the foreign DNA

Key Terms

• DNA cloning • Set of procedures that uses living cells to make many identical copies

of a DNA fragment

• plasmid• Of many bacteria and archaeans, a small ring of nonchromosomal

DNA replicated independently of the chromosome

• cloning vector • A DNA molecule that can accept foreign DNA, be transferred to a host

cell, and get replicated in it

Plasmid Cloning Vectors

Fig. 15.3, p. 220

Kpn l

Sph l

Pst l

Bam Hl

Eco RI

Sal l

Acc l

Xho l

Xba l

Bst XI

Sac l

Not l

pDrive Cloning Vector3.85 kb

BA

Plasmid Cloning Vectors

Fig. 15.3a, p. 220

Plasmid Cloning Vectors

Fig. 15.3b, p. 220

Plasmid Cloning Vectors

DNA Cloning

Fig. 15.4, p. 221

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

cutplasmid

recombinant plasmid

plasmid cloning vector

chromosomalDNA fragmentschromosomal

DNA

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

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

DNA Cloning

Fig. 15.4, p. 221

recombinant plasmid

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

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

DNA Cloning

ANIMATION: Formation of recombinant DNA

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cDNA Cloning

• Researchers who study eukaryotic genes and their expression work with mRNA transcripts of genes

• RNA can’t be cloned directly – reverse transcriptase, a viral enzyme, is used to transcribe single-stranded RNA into complementary DNA (cDNA) for cloning

Key Terms

• reverse transcriptase • A viral enzyme that uses mRNA as a template to make a

strand of cDNA

• cDNA • DNA synthesized from an RNA template by the enzyme

reverse transcriptase

cDNA Cloning (1)

• A strand of cDNA, is assembled on an mRNA template:

cDNA Cloning (2)

• DNA polymerase removes RNA and copies the cDNA into a second strand of DNA, resulting in a double-stranded DNA copy of the original mRNA:

Key Concepts

• DNA Cloning• Researchers routinely make recombinant DNA by cutting

and pasting together DNA from different species• Plasmids and other vectors can carry foreign DNA into

host cells

ANIMATION: Base-pairing of DNA fragments

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ANIMATION: How to Make cDNA

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15.3 From Haystacks to Needles

• DNA libraries are sets of cells that host cloned DNA fragments• A genomic library collectively contains all DNA in a

genome• A cDNA library contains only those genes being expressed

at the time the mRNA was harvested

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

Key Terms

• DNA library • Collection of cells that host different fragments of foreign

DNA, often representing an organism’s entire genome

• genome • An organism’s complete set of genetic material

Isolating Genes

• Researchers use probes that match a targeted DNA sequence to identify cells with a specific DNA fragment

• A probe mixed with DNA from a library base-pairs with (hybridizes to) the targeted gene

• Base pairing between nucleic acids from different sources is called nucleic acid hybridization

Key Terms

• probe • Short fragment of DNA labeled with a tracer such as a

radioactive isotope• Designed to hybridize with a nucleotide sequence of

interest

• nucleic acid hybridization • Base-pairing between DNA or RNA from different sources

Nucleic Acid Hybridization

Fig. 15.5, p. 222

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.

Nucleic Acid Hybridization

ANIMATION: Use of a radioactive probe

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PCR

• The polymerase chain reaction (PCR) uses primers and heat-resistant DNA polymerase to mass-produce a particular section of DNA without having to clone it in living cells

• polymerase chain reaction (PCR) • Method that rapidly generates many copies of a specific

section of DNA

• primer • Short, single strand of DNA designed to hybridize with a

DNA fragment

Multiplication by PCR

• PCR can be used on any sample of DNA with at least one molecule of a target sequence

• Essentially any sample containing DNA can be used, even one sperm, a hair left at a crime scene, or a mummy

• Each cycle of a PCR reaction doubles the number of copies of a section of DNA – thirty cycles can make a billion copies

Two Rounds of PCR (1)

• DNA template is mixed with primers, nucleotides, and heat-tolerant Taq DNA polymerase

Fig. 15.6.1, p. 223

targeted section DNA template (blue) is mixed with primers (pink), nucleotides, and heat-tolerant Taq DNA polymerase.

1

Two Rounds of PCR (1)

Two Rounds of PCR (2)

• When the mixture is heated, the double-stranded DNA template separates into single strands – when it is cooled, primers base-pair with template DNA

Fig. 15.6.2, p. 223

When the mixture is heated, the double-stranded DNA template separates into single strands. When it is cooled, some of the primers base-pair with the template DNA.

2

Two Rounds of PCR (2)

Two Rounds of PCR (3)

• Taq polymerase begins DNA synthesis at primers, so complementary DNA strands form on single-stranded templates

Fig. 15.6.3, p. 223

Taq polymerase begins DNA synthesis at the primers, so complementary strands of DNA form on the single-stranded templates.

3

Two Rounds of PCR (3)

Two Rounds of PCR (4)

• The mixture is heated again; double-stranded DNA separates into single strands

• When it is cooled, primers basepair with old and new DNA strands

Fig. 15.6.4, p. 223

The mixture is heated again, and the double-stranded DNA separatesinto single strands. When it is cooled, some of the primers base-pairwith the template DNA. The copied DNA also serves as a template.

4

Two Rounds of PCR (4)

Two Rounds of PCR (5)

• Each round of PCR reactions doubles the number of copies of the targeted DNA section

Fig. 15.6.5, p. 223

Each round of PCR reactions can double the number of copies of the targeted DNA section.5

Two Rounds of PCR (5)

ANIMATION: Polymerase chain reaction (PCR)

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Key Concepts

• Finding Needles in Haystacks• DNA libraries, hybridization, and PCR are techniques that

allow researchers to isolate and make many copies of a fragment of DNA they want to study

ANIMATION: Polymerase chain reaction

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15.4 DNA Sequencing

• DNA sequencing reveals the order of bases in DNA

• The entire genomes of several organisms have now been sequenced

• DNA sequencing• Method of determining the order of nucleotides in DNA• DNA polymerase partially replicates a DNA template• Produces a mixture of DNA fragments of different lengths• Fragments are separated by electrophoresis

Electrophoresis

• Electrophoresis separates fragments by length into bands • Electric field pulls DNA fragments through semisolid gel• Fragments of different sizes move at different rates• Shorter fragments move through the gel faster than longer

fragments do

• electrophoresis • Technique that separates DNA fragments by size

5 Steps in DNA Sequencing1. Sequencing depends on dideoxynucleotides to terminate DNA

replication – each is labeled with a colored pigment

Fig. 15.7.1, p. 224

dideoxynucleotides

1

5 Steps in DNA Sequencing

5 Steps in DNA Sequencing

2. DNA polymerase uses a section of DNA as a template to synthesize new strands of DNA – synthesis of each new strand stops when a dideoxynucleotide with a tracer is added

3. At the end of the reaction, the mixture contains many incomplete copies of the original DNA

5 Steps in DNA Sequencing

4. Electrophoresis separates DNA fragments into bands according to length – all DNA strands in each band end with the same dideoxynucleotide, and are the same color

5. A computer detects and records the color of successive bands on the gel – the order of colors represents the sequence of the template DNA

DNA Sequencing

Fig. 15.7.2-5, p. 224

DNA template

2

3

4

5

DNA Sequencing

3D Animation: Gene Sequencing

The Human Genome Project

• The human genome consists of about 3 billion bases

• Hoping to patent the sequence, Craig Venter’s company, Celera Genomics, invented faster methods of sequencing genomic DNA using supercomputers

• By 2003, the human genome sequence was completed – but governments decided the sequence could not be patented

Human Genome Sequencing

Key Concepts

• DNA Sequencing• Sequencing reveals the linear order of nucleotides in DNA• Comparing genomes offers insights into human genes and

evolution• An individual can be identified by unique parts of their

DNA