Title: Figure 20.2 Overview of gene cloning
1Figure 20.2 Overview of gene cloning
2Chapter 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
3Chapter 20 DNA Technology and Genomics
- How is a gene cut out of a chromosome?
- How is recombinant DNA cloned?
4Chapter 20 DNA Technology and Genomics
- How is a gene cut out of a chromosome?
- How is recombinant DNA cloned?
5Chapter 20 DNA Technology and Genomics
- How is a gene cut out of a chromosome?
- How is recombinant DNA cloned?
6Chapter 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
7Chapter 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
8Figure 20.5 Nucleic acid probe hybridization
9Chapter 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!
10Chapter 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
11Figure 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
12Chapter 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.
13Figure 20.8 Gel Electrophoresis
- DNA loaded into wells
- Electrical current applied
- (-) DNA moves toward ()
- Shorter molecules move faster
- DNA is visualized
14Chapter 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
15Figure 20.9 Using restriction fragment analysis
to distinguish the normal and sickle-cell alleles
of the ?-globin gene
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17- Crime Scene DNA (RFLP) O.J. Simpson
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19RFLP 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)
20RFLP 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
21Chapter 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)
22Figure 20.10 Southern blotting of DNA fragments
23Chapter 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?
24Chapter 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.
25Chapter 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
26Figure 20.14 Research Method DNA microarray assay
of gene expression levels
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28- Genetically-modified crops!!!
- Pharm Animals!!!
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31DNA 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.
32Lab 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.