Chapter 5: Exploring Genes and Genomes

Biotechnology/Medicine

Diagnosis Treatment Identification Crime .

Techniques:
1. 2. 3. 4. 5. 6. 7. 8. 9. Restriction enzymes (1978, Nobel prize) Blotting techniques, Microarray (2005, Lasker award) DNA sequencing (1980, Nobel prize) Molecular cloning (1980, Nobel prize) Solid phase oligonucleotide synthesis (1984, Nobel prize) PCR (1993, Nobel prize) RNA interference (2006, Nobel prize) Mutagenesis (1993, Nobel prize) Transgenic/knockout animal (homologous recombination) (2007, Nobel prize)

1. Restriction enzyme:
found in prokaryotes to cut foreign (viral) DNA.

strain number E.coli

Recognize palindromic sequence Hydrolyze both strands

DNA Restriction Analysis:

Mapping of restriction enzyme cleavage sites within a DNA sequence. 1. cut DNA with restriction enzymes 2. size fractionate fragments by agarose or polyacrylamide (for smaller fragments) gel electrophoresis 3. detect bands by ethidium bromide staining

Example: EcoRV restriction enzyme

Cut

Cut

Example: EcoRV restriction enzyme Enzyme Dimer

DNA

Gel electrophoresis pattern of a restriction digest

(SV40 DNA cut with three different restriction enzymes)

compare patterns ~ polymorphism, disease diagnosis molecular cloning

2. Blotting Techniques (detection of genes or proteins):

Southern blot : DNA (template) DNA (probe) hybridization Northern blot : RNA DNA hybridization Western blot : Protein - Antibody binding
4. Transfer to nitrocellulose membrane 5. Hybridize nitrocellulose with radiolabeled probe 6. Wash and autoradiograph

Denature Single strand

3. Sequencing of DNA:
Determination of the order of nucleotide sequence within a DNA fragment
1. Requires template strand (single strand) and a specific primer 2. Dideoxynucleotides, which lack 3 OH, are used to cause chain termination 3. A radioactive or fluorescent nucleotide is included so that the elongated primer strand is radioactive or fluorescent labeled. 4. The reaction products of four separate reactions are size fractionated by acrylamide gel electrophoresis 5. Sequence of the DNA is read from bottom (lower molecular weight) to top

Dideoxynucleotides, which lack 3 OH, cause chain termination

DNA polymerization

Sequencing results
Radioisotope ~autoradiography
longer

Fluorescence detection

shorter read

(automated---genome center)

4. Cloning DNA fragments (Recombinant DNA technology)

Cloning requires a vehicle (carrier) for replication of DNA. The vehicle (vector) can be: 1. a plasmid 2. a bacteriophage 3. a virus 4. a whole genome The vehicle is a DNA sequence capable of replication.

The DNA to be cloned is ligated to vehicle

The DNA is then amplified along with vehicle

Examples of Vectors
(i) Plasmid: works as an extra chromosome in bacteria)

insert

(ii) Virus:

1. Cut plasmid with restriction enzymes 2. Cut DNA to be cloned with compatible restriction enzymes 3. Ligate plasmid and DNA fragment using DNA ligase enzyme

vector

insert

(cohesive)

4. Transform bacteria, select transformants (i) with antibiotic resistance, or (ii) by colony hybridization 5. Grow bacteria and isolate plasmid

(i) antibiotic resistance

5. Synthesis of DNA using protected nucleoside phosphoramidites

(amino groups)

Solid-phase synthesis of a DNA chain by the phosphite triester method 5 3

(no polymerase)

Used to create primers for sequencing, PCR Now possible to synthesize genes

6. Polymerase Chain Reaction (PCR)

A. Template DNA B. Synthetic oligonucleotide primers C. Thermostable DNA polymerases (Taq polymerase - isolated from bacteria from hot springs) D. dNTPs dATP, dGTP, dCTP, dTTP E. Thermal cycling devices

~94C

37~70C
Sequences need to be known Specificity of PCR can be controlled by: annealing temperature salt concentration, etc.

3 5

5 3

~70C 3 3 5 5 5 5 3 3

PCR

5 5 5

5
5 5 3

3 5

Short strands

* *

PCR

5
5

* *
5

* *
5 5

*
5

*
5

2n

PCR Movie

PCR - Applications to medical diagnosis

DNA isolated from patients blood is PCR amplified to test for immunodeficiency virus, tuberculosis, translocation, etc.

PCR Applications to forensics

DNA isolated from blood stains on the jeans and shirt from defendant is PCR amplified (HLA) and then compared with DNA from the victim (V) and defendant (D)

(blood on Ds)

HLA: human leukocyte antigen Very diverse in human population; the chance of two unrelated individuals having identical HLA molecules is very low.
Short Tandem Repeats (STR) Repeated sequences come in various sizes (size unique to individual; unknown function).

7. RNA interference
A tool for disrupting gene expression: Specific double stranded RNA (dsRNA), which contains complementary sequences to the target gene, is introduced into a cell, and suppresses the transcription of the target gene. dsRNA is cleaved by Dicer to produce small interfering RNAs (siRNAs). siRNAs gets incorporated into the RNA induced silencing complex (RISC), where the single-stranded RNAs guide the cleavage of mRNAs containing complementary sequences. --> cancer therapy etc.
Cell
(Ribonuclease)

(21bp)

8. Oligonucleotidedirected mutagenesis.
A method to produce a desired change in the DNA sequence. DNA sequence must be known and must be cloned into a vector. Design a complimentary primer with a base substituted at the desired place, fill in the remaining bases using DNA polymerase, close ends using ligase, separate the newly synthesized DNA strand from the template strand and amplify the new DNA which will contain the single point mutation.

DNA synthesis

9. Sequencing of the Human Genome

Finished sequence reported in 2004. Total ~25,000 genes that encode for proteins. Three billion base pairs.

(46 chromosomes - 22 pairs of autosomes and the X and Y sex chromosomes)

Genome comparison

Share 99% of genes Large chromosomal fragments have re-assorted

10. High throughput/Next Generation/deep sequencing technology

Can sequence >10 billion bases of DNA in one experiment. Widespread whole genome sequencing for diagnostic or preventive medical testing is likely within the foreseeable future. Diagnosis of genetic diseases Screening for genetic risk factors Identification of mutations in tumor cells Forensic identification Identification of pathogens (viruses, bacteria, etc) Generates short sequences (typically 50-150 bases) that are then mapped to a known genome (can be computationally intensive) Some challenges: Analysis costs and automation of analysis process Interpretation of variations/mutations (knowledge base) Which sequence variations have biological consequences? Ethics and privacy concerns

10. Transgenic mice

Mice engineered to carry foreign genes as part of its own genetic material
Microinjection

Transgenic mice

+GH DNA construct

~ gene therapy

11. Gene disruption by homologous recombination

Homologous recombination: important for replication, repair, diversity

: genome

=
: construct in Embryonic Stem cell

Generation of knockout mice

Blastocysts are implanted into foster mothers to become embryos

Targeted ES cells are injected into blastocysts (fertilized egg before implantation)

female (normal) Give birth to chimeric mice male (chimera) Sperm Gene targeted mice
(normal mice)

Mating between chimeric mouse and normal mouse

--> Knockout mice - to know gene function in vivo

(from Medgadget)

Muscle atrophy in myogenin knockout mice

Normal mouse Knockout mouse

Muscles

Summary
Restriction Enzymes Electrophoresis Blotting DNA Sequencing Cloning DNA Solid phase synthesis of DNA PCR Mutagenesis RNA interference Transgenic and Knockout animals