Albia Dugger Miami Dade College Cecie Starr Christine Evers Lisa Starr Chapter 15 Biotechnology (Sections )

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 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 Vector 3.85 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. cut plasmid recombinant plasmid plasmid cloning vector chromosomal DNA fragments chromosomal 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. cut plasmid chromosomal DNA 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 To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERECLICK HERE

cDNA Cloning Researchers who study eukaryotic genes and their expression work with mRNA transcripts of genes (not the DNA itself, because DNA has sequences that are not part of genes – “introns”. In mRNA, the introns have been cut out, and all that is left is the sequence of the needed gene.) 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 To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERECLICK HERE

ANIMATION: How to Make cDNA To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERECLICK HERE

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

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 , 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 , 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 , 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 , p. 223 The mixture is heated again, and the double-stranded DNA separates into single strands. When it is cooled, some of the primers base-pair with 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 , 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) To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERECLICK HERE

Key Concepts Finding Needles in Haystacks DNA libraries 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 To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERECLICK HERE

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

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