Post on 11-Jan-2016
Chapter 20DNA Technology and Genomics
•DNA cloning
•DNA probe hybridization
•Polymerase Chain Reaction (PCR)
•Gel Electrophoresis
•Southern Blot
•DNA sequencing.
Chapter 20DNA Technology and Genomics
Bacteria have restriction enzymes to attack and destroy invading viral DNA.
Restriction enzymes cut DNA at specific nucleotide sequences leaving “sticky ends.”
DNA ligase can seal these ends, making recombinant DNA.
Chapter 20DNA Cloning
Restriction fragments can be put into plasmids.
Gene cloning occurs when cells containing these plasmids reproduce.
Genes of interest are marked with a radioactive DNA probe.
Chapter 20DNA Cloning
If a gene is inserted next to a promoter, the bacteria becomes an expression vector.
Eukaryotic chromosomes allow for bigger segments of DNA.
Eukaryotic cells can also process polypeptides into proteins.
Chapter 20DNA Cloning
Chopping up the whole genome of an organism produces many DNA fragments containing many genes.
Often, the researcher will save all of them, either in bacteria or in viruses.
These collections of bacterial clones are called genomic libraries.
Chapter 20DNA Cloning
Chapter 20DNA Probe Hybridization
Samples of DNA
Chapter 20DNA Probe Hybridization
Heat to 95o to denature
Chapter 20DNA Probe Hybridization
Introduce radioactive probe
Chapter 20DNA Probe Hybridization
Allow to cool
Chapter 20DNA Probe Hybridization
Measure radioactivity
√ x x
Polymerase chain reaction (PCR) uses DNA polymerase to clone DNA in vitro.
In vitro = in a test tube
In vivo = in a living organism
Chapter 20Polymerase Chain Reaction (PCR)
DNA FingerprintingHuman DNA contains lots of noncoding sequences that serve no purpose.
This “junk DNA” often repeats over and over.
No two people (except identical twins) have exactly the same repeats.
Chapter 20Gel Electrophoresis
DNA Fingerprinting
Bill’s chromosome:gene A xxxxx gene B yyy gene C
Bob’s chromosome:gene A xx gene B yyyyy gene C
Chapter 20Gel Electrophoresis
DNA FingerprintingRestriction enzymes cut DNA at specific places.
Bill’s chromosome:gene A xxxxx gene B yyy gene C
Bob’s chromosome:gene A xx gene B yyyyy gene C
Chapter 20Gel Electrophoresis
DNA FingerprintingRestriction enzymes cut DNA at specific places.
Bill’s chromosome:gene A xxxxx gene B yyy gene C
Bob’s chromosome:gene A xx gene B yyyyy gene C
Chapter 20Gel Electrophoresis
DNA FingerprintingRestriction enzymes cut DNA at specific places.
Bill’s chromosome:gene A xxxxx gene B yyy gene C
Bob’s chromosome:gene A xx gene B yyyyy gene C
Chapter 20Gel Electrophoresis
DNA FingerprintingRestriction enzymes cut DNA at specific places.
Bill’s chromosome:
Bob’s chromosome:
Chapter 20Gel Electrophoresis
DNA FingerprintingLonger fragments travel more slowly through the gel.
Bill’s chromosome:
Bob’s chromosome:
Chapter 20Gel Electrophoresis
DNA FingerprintingBill: Bob:
Chapter 20Gel Electrophoresis
DNA FingerprintingBill: Bob:
Chapter 20Gel Electrophoresis
DNA FingerprintingBill: Bob:
Chapter 20Gel Electrophoresis
DNA FingerprintingBill: Bob:
Chapter 20Gel Electrophoresis
Chapter 20Gel Electrophoresis
The southern blot
1.Do DNA fingerprinting on an entire genome.
2.Blot the DNA from the gel to paper with an alkaline solution. This denatures the DNA.
3.Hybridize with a radioactive probe.
Chapter 20Southern Blot
The southern blot
Chapter 20Southern Blot
The southern blot
Chapter 20Southern Blot
Chapter 20DNA Sequencing
1. Synthesize DNA 3’ → 5’
2. Stop synthesis at random
3. Dideoxyribonuceotides with dye
4. Separate fragments by length
5. Read the colors of the lengths.
Chapter 20DNA Sequencing
Synthesize DNA 3’ → 5’
Chapter 20DNA Sequencing
Synthesize DNA 3’ → 5’
5-AGTACCTG-3
Chapter 20DNA Sequencing
Synthesize DNA 3’ → 5’
5-AGTACCTG-3 3-TCATGGAC-5
Chapter 20DNA Sequencing
Stop the process with didexoyribonucleotides
5-AGTACCTG-3 3-TCATGGAC-5
Chapter 20DNA Sequencing
Stop the process with didexoyribonucleotides
5-AGTACCTG-3 3-TCATG
Chapter 20DNA Sequencing
Add a different dye to each didexoyribonucleotide
A T C G
5-AGTACCTG-3
Chapter 20DNA Sequencing
Stop the synthesis at random times with different dyes
A T C G
5-AGTACCTG-3
Chapter 20DNA Sequencing
Stop the synthesis at random times with different dyes
5-AGTACCTG-3 3-TCATGG
Chapter 20DNA Sequencing
Stop the synthesis at random times with different dyes
5-AGTACCTG-3 3-TCATGG 3-T
Chapter 20DNA Sequencing
Stop the synthesis at random times with different dyes
5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC
Chapter 20DNA Sequencing
Stop the synthesis at random times with different dyes
5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA
Chapter 20DNA Sequencing
Stop the synthesis at random times with different dyes
5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA 3-TCATG
Chapter 20DNA Sequencing
Stop the synthesis at random times with different dyes
5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA 3-TCATG 3-TCA T
Chapter 20DNA Sequencing
5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA 3-TCATG 3-TCA T
Arrange the fragments by length
Chapter 20DNA Sequencing
3-T 3-TC 3-TCA 3-TCA T 3-TCATG 3-TCATGG 3-TCATGGA3-TCATGGAC
Arrange the fragments by length
Chapter 20DNA Sequencing
3-T 3-TC 3-TCA 3-TCA T 3-TCATG 3-TCATGG 3-TCATGGA3-TCATGGAC
Read the colors
Chapter 20DNA Sequencing
Chapter 20DNA Sequencing
RFLPs (“RIF-lips”), or restriction fragment length polymorphisms, are differences in homologous chromosomes that give different length restriction fragments.
Chromosome walking means finding where fragments of DNA overlapped in the genome.
Chapter 20DNA Technology and Genomics
Genomics is the systematic study of entire genomes.
Proteomics is the study of all the proteins encoded by a genome.
Single nucleotide polymorphisms (SNPs) are useful markers for studying variation.
Chapter 20DNA Technology and Genomics
Uses of DNA Technology:
Testing for genetic diseases
Large scale production of drugs
Gene therapy
Forensics
Genetic engineering.
.
Chapter 20DNA Technology and Genomics