Figure 20.2 Overview of gene cloning

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Figure 20.2 Overview of gene cloning
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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

3
Chapter 20 DNA Technology and Genomics

1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?

4
Chapter 20 DNA Technology and Genomics

1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?

5
Chapter 20 DNA Technology and Genomics

1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?

6
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

7
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

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Figure 20.5 Nucleic acid probe hybridization
9
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 importantno INTRONS, so can
  be directly
• inserted into bacteria for protein
  production!

10
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

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Figure 20.7 The polymerase chain reaction (PCR)

• Making DNA
• Template
• Primers
• dNTPs (free nucleotidesA, T, C, G)
• DNA polymerase

(Taq heat resistant)

• Denature DNA 95C
• Annealing allow primers to bind
• Extension polymerase builds new DNA
• Repeat this cycle 25 35 times
• Each cycle doubles the DNA

12
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.

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Figure 20.8 Gel Electrophoresis

1. DNA loaded into wells
2. Electrical current applied
3. (-) DNA moves toward ()
4. Shorter molecules move faster
5. DNA is visualized

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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

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Figure 20.9 Using restriction fragment analysis
to distinguish the normal and sickle-cell alleles
of the ?-globin gene
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• Crime Scene DNA (RFLP) O.J. Simpson

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RFLP Somewhat outdated. Whats next?
-RFLP cant 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)
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RFLP Somewhat outdated. Whats 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
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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)

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Figure 20.10 Southern blotting of DNA fragments
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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?

24
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.

25
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

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Figure 20.14 Research Method DNA microarray assay
of gene expression levels
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• Genetically-modified crops!!!
• Pharm Animals!!!

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• Gene Therapy

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DNA Forensics Lab

• On the back of your lab packet
• 1) According to your evidence, which suspects
  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.

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Lab Unit Test3/20/15

• 1) Put Learning Logs in blue bin.
• 2) Put DNA Forensics Lab Packet in pile beside
  blue bin (if you didnt 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.