Figure 20.2 Overview of gene cloning Bacterium Bacterial chromosome Plasmid Cell containing gene of interest Gene inserted into plasmid Recombinant DNA (plasmid) Plasmid put into bacterial cell Gene of interest DNA of chromosome Recombinate bacterium Host cell grown in culture, to form a clone of cells containing the “cloned” gene of interest Protein harvested Basic research on protein Basic research and various applications Copies of gene Basic research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth Protein expressed by gene of interest 1 2 3 4

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? Restriction enzymes Recognize a palindrome sequence Originally found in bacteria Overhangs are “sticky ends” & will bind to any complementary sequence DNA ligase makes a recombinant DNA molecule Restriction site DNA 5 3 G A A T T C C T T A A G Restriction enzyme cuts the sugar-phosphate backbones at each arrow DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. DNA ligase seals the strands. Sticky end Fragment from different DNA molecule cut by the same restriction enzyme One possible combination Recombinant DNA molecule G C T T A A A A T T C A A T T C T T A A 1 2 3

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? Recombinant DNA plasmids Sticky ends Human DNA Fragments Human cell Gene of interest Bacterial cell ampR gene (ampicillin resistance) Bacterial plasmid Restriction site lacZ gene (lactose breakdown) 1 Isolate plasmid DNA and human DNA. 2 Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA. 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids.

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? Recombinant bacteria Recombinant DNA plasmids Sticky ends Human DNA Fragments Human cell Gene of interest Bacterial cell ampR gene (ampicillin resistance) Bacterial plasmid Restriction site lacZ gene (lactose breakdown) 1 Isolate plasmid DNA and human DNA. 2 Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA. 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. 4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene.

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone Colony carrying re- combinant plasmid with disrupted lacZ gene Recombinant bacteria Recombinant DNA plasmids Sticky ends Human DNA Fragments Human cell Gene of interest Bacterial cell ampR gene (ampicillin resistance) Bacterial plasmid Restriction site lacZ gene (lactose breakdown) 1 Isolate plasmid DNA and human DNA. 2 Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA. 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. 4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. 5 Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow.

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? Genomic libraries Collection of clones in either plasmids or phages Foreign genome cut up with restriction enzyme Recombinant plasmids Recombinant phage DNA Phage clones (b) Phage library (a) Plasmid library or Bacterial clones

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? Screen a genomic library using a radioactive probe Nucleic acid probe hybridization

Figure 20.5 Nucleic acid probe hybridization

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? complementary DNA complementary to processed mRNA Only exons present Isolate mRNA Use reverse transcriptase to make cDNA cDNA libraries are important…no INTRONS, so can be directly inserted into bacteria for protein production!

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? Polymerase chain reaction Used to amplify DNA Forensics Paternity testing To aid in DNA sequencing

Figure 20.7 The polymerase chain reaction (PCR) Making DNA Template Primers dNTPs (free nucleotides—A, T, C, G) DNA polymerase (Taq – heat resistant) Denature DNA – 95°C Annealing – allow primers to bind Extension – polymerase builds new DNA Repeat this cycle 25 – 35 times Each cycle doubles the DNA

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? Method to separate DNA or protein based on size & charge Forest analogy….

Figure 20.8 Gel Electrophoresis DNA loaded into wells Electrical current applied (-) DNA moves toward (+) Shorter molecules move faster DNA is visualized

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? What is RFLP analysis? Restriction Fragment Length Polymorphism Combines restriction digest & gel electrophoresis

Figure 20.9 Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the -globin gene Normal  -globin allele Sickle-cell mutant -globin allele 175 bp 201 bp Large fragment DdeI Ddel 376 bp DdeI restriction sites in normal and sickle-cell alleles of -globin gene. Electrophoresis of restriction fragments from normal and sickle-cell alleles. Normal allele Sickle-cell allele 201 bp 175 bp (a) (b)

Crime Scene DNA (RFLP): O.J. Simpson

RFLP: Somewhat outdated. What’s next? -RFLP can’t be used for degraded DNA or very small amounts of DNA, so… -STR (short tandem repeats) analysis has replaced RFLP in forensic science. -Look at both parental versions of known STRs in DNA and COUNT how many repeats exist.  Compare suspect with crime scene evidence for match! LOCUS BELOW would be designated (7, 8)

RFLP: Somewhat outdated. What’s next? -The more loci are compared, the more accurate the results.  Comparisons at 13 loci are sufficient to positively ID a suspect. CODIS: Combined DNA Index System

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? What is RFLP analysis? What is Southern blot analysis? Combination of RFLP & nucleic acid probe hybridization Transfers DNA from gel to a solid substrate (nitrocellulose paper)

Figure 20.10 Southern blotting of DNA fragments

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? What is RFLP analysis? What is Southern blot analysis?

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? What is RFLP analysis? What is Southern blot analysis? How can gene function be determined? in vitro mutagenesis – disable gene & observe consequences RNA interference (RNAi) – silencing of gene expression by using miRNA/siRNA with matching sequence which triggers breakdown of mRNA.

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? What is RFLP analysis? What is Southern blot analysis? How can gene function be determined? in vitro mutagenesis – disable gene & observe consequences RNA interference (RNAi) – silencing of gene expression by using DS-RNA with matching sequence which triggers breakdown of mRNA. 11. What is a DNA microarray? Method used to measure expression of thousands of genes at once

Figure 20.14 Research Method DNA microarray assay of gene expression levels

Genetically-modified crops!!! “Pharm” Animals!!!

Gene Therapy

DNA Forensics Lab On the back of your lab packet… 1) According to your evidence, which suspect’s DNA matched the DNA found at the crime scene? 2) Draw your gel, with all 6 lanes labeled. 3) Thoroughly explain the process that you utilized to attain your results. Within your answer, describe the PCR process, describe the gel electrophoresis process, and discuss the reasons that these two processes were necessary for your investigation.

Lab Unit Test—3/20/15 1) Put Learning Logs in blue bin. 2) Put DNA Forensics Lab Packet in pile beside blue bin (if you didn’t submit it yesterday.) 3) Take a scantron (BLUE SIDE) from the pile. Mark KEY ID (A or B) 4) Put your name on everything except the released AP exam question sheet. 5) Cans due by the end of next week. Meet back in my room on Monday.