DNA Technology

1
DNA Technology

• Monday, August 11

2
Overview

• Biotechnology manipulation of organisms or
  their components to make useful products
• Genetic engineering direct manipulation of
  genes for practical purposes
• Recombinant DNA genes from two different
  sources are combined in vitro into the same
  molecule

3
DNA Cloning

• How do you target a gene to study it?
• 1/100,000 of a chromosome
• Only differ in nucleotide sequence
• Gene cloning
• Well-defined, gene-sized pieces of DNA in
  multiple identical copies
• Genes are isolated and inserted into plasmids
• Small, circular pieces of DNA that replicate in
  bacteria cells independently of chromosomes

4
Figure 20.1 An overview of how bacterial
plasmids are used to clone genes
Gene gives organism a new metabolic property
5
How do you cut and paste DNA?

• Enzymes that cut DNA at specific short sequence
  sites
• Restriction enzymes digest DNA
• Blunt end cut
• Asymmetric end cut
• Enzymes that paste complementary DNA fragments
  together
• DNA ligase

6
Figure 20.2 Using a restriction enzyme and DNA
ligase to make recombinant DNA
7
Figure 20.3 Cloning a human gene in a bacterial
plasmid a closer look

• Plasmid cloning vector
• AmpR confers resistance to the antibiotic
  ampicillin
• Only bacteria with this gene can grow in the
  presence of ampicillin
• lacZ catalysis hydrolysis of lactose
• Reaction in cells turns them blue
• Restriction site in the middle of the lacZ gene

8
Figure 20.3 Cloning a human gene in a bacterial
plasmid a closer look
Note Human gene has inserted into the middle of
the lacZ gene, disrupting it
9
Figure 20.3 Cloning a human gene in a bacterial
plasmid a closer look
Transformation the uptake of naked DNA from the
surrounding solution
10
Figure 20.3 Cloning a human gene in a bacterial
plasmid a closer look
Bacterial colonies Any colony that grows
must be resistant to amp - they must have the
ampR gene and thus contain the plasmid Blue
colonies have functional lacZ, so do not have
inserted gene White colonies do not have lacZ
so they have inserted gene
11
Figure 20.3 Cloning a human gene in a bacterial
plasmid a closer look
Identifying clones of interest Nucleic acid
hybridization Nucleic acid probe short,
single-stranded segment of DNA complementary to
part of the gene sequence Probe binds gene and
detect with tag (fluorescence or
radioactivity) Detect presence of protein
product of the gene Activity or structure
12
Figure 20.4 Using a nucleic acid probe to
identify a cloned gene
13
Expressing Eukaryotic Genes

• Promoter differences expression vector
• Prokaryotic promoter upstream of a restriction
  site where the eukaryotic gene can be inserted
• Eukaryotic genes have introns
• Utilize splice mRNA from cells and reverse
  transcribe into DNA
• Eukaryotic proteins are post-translationally
  modified
• Eukaryotic cells as cloning hosts
• Yeast artificial chromosomes

14
Figure 20.5 Making complementary DNA (cDNA) for
a eukaryotic gene
15
Figure 20.6 Genomic libraries a complete set
of recombinant clones carrying copies of DNA
segments from the entire genome
cDNA library A collection of all the mRNA made
in a cell (set of all transcribed genes)
16
The polymerase chain reaction (PCR)

• Fast way to amplify specific piece of DNA from
  small amounts of starting material
• In vitro transcription
• What do you need?

17
The polymerase chain reaction (PCR)

• Fast way to amplify any piece of DNA
• In vitro transcription
• What do you need?
• Enzyme DNA polymerase
• Nucleotides (A, C, T, G)
• DNA Template
• Primers (DNA pol can only add bases to a
  preexisting chain)
• Specify the DNA to be amplified

18
Figure 20.7 The polymerase chain reaction (PCR)
1. Denature
2. Anneal
3. Extension
19
DNA Analysis

• Restriction Fragment Analysis indirectly
  detects certain differences in the nucleotide
  sequences of DNA molecules
• Gel Electrophoresis
• Separates macromolecules (DNA or protein) on the
  basis of size and/or charge
• Southern Blotting
• Detection of all DNA molecules containing a
  specific sequence by nucleic acid probe
  hybridization

20
Figure 20.8 Gel electrophoresis of macromolecules
The rate of DNA movement is related to its size
the longer the fragment DNA the slower it moves
The phosphates in the sugar-phosphate backbone of
DNA gives it a negative charge
DNA fragments are arrayed in bands along a lane
according to size
21
Figure 20.9 Using restriction fragment patterns
to distinguish DNA from different alleles
A DNA-binding dye allows visualization of the DNA
bands under UV light
22
Figure 20.x1a Laboratory worker reviewing DNA
band pattern
23
Figure 20.10 Restriction fragment analysis by
Southern blotting
Characteristic pattern of bands for each sample
DNA is transferred to paper and denature to
single strands
Entire genome
Probe complementary to the DNA sequence of
interest
DNA bound to radioactive probe exposes film
24
Genomics

• Studying the entire genome sequence of an
  organism
• Human Genome Project (begun 1990)
• Determining the complete nucleotide sequence of
  the DNA of each human chromosome
• Genetic mapping
• Physical mapping
• DNA sequencing

25
Figure 20.13 Alternative strategies for
sequencing an entire genome
26
Figure 20.12 Sequencing of DNA by the Sanger
method
27
Figure 20.12 Sequencing of DNA by the Sanger
method
ddNTP dideoxyribonucleotide
Added to the end of a growing chain and
terminates synthesis
28
Figure 20.12 Sequencing of DNA by the Sanger
method
The DNA strands of various lengths (differing by
one nucleotide) are separated
29
Figure 20.12 Sequencing of DNA by the Sanger
method
30
Table 20.1 Genome Sizes and Numbers of Genes
Surprisingly small number of genes ! Unusually
large amount of noncoding DNA Diversity comes
from mixing and matching modular elements (exons
and protein domains)
31
Studying Gene Expression

• We now have methods to obtain genes and sequence
  them
• We can use this information to learn about how
  interactions between genes and their products run
  a cell
• Determine structure from sequence
• Study patterns of gene expression
• Which genes are transcribed in different
  situations
• mRNA

32
Figure 20.14a DNA microarray assay for gene
expression
33
Figure 20.14b DNA microarray assay for gene
expression
34
Studying Gene Function

• In vitro mutagenesis
• Manipulate DNA to alter or destroy functions
• Promoter bashing
• Protein destruction
• Proteomics
• Systematic study of the full protein sets encoded
  by genomes
• Bioinformatics
• Application of computer science and mathematics
  to genetic and other biological information

35
Applications of DNA Technology

• Diagnosis of disease
• Viral genome detection (HIV)
• Genetic disorders (screen for defective genes
  hemophilia, CF, breast cancer)
• Production of pharmaceutical products
• Insulin for diabetes
• Gene Therapy
• Replace or supplement of a defective gene

36
Figure 20.16 One type of gene therapy procedure
Retrovirus insert their DNA along with the normal
gene into the genome of the patient cells
Bone marrow, stem cells multiply throughout the
patients life
37
Other applications.

• Environmental Uses
• Mining minerals
• Detoxifying wastes (oil, sewage, pollution)
• Agricultural Uses
• Transgenic organisms
• Sheep with better wool
• Pig with leaner meat
• Genetic engineering in plants
• Resistant to disease and spoilage
• Delayed ripening
• Forensic Investigation
• Identifying criminal by DNA fingerprinting
• Paternity tests

38
Figure 20.17 DNA fingerprints from a murder case
PCR amplify small amounts of DNA from crime
scene Digest DNA and compare pattern of bands
DNA fingerprint
39
Ethical Issues

• Genetically modified organisms
• Artificially altering global organization of life
• Environmental and health issues
• Designer kids
• Identifying disease genes
• Health care
• Employment