Slide 1

DNA Technology 2
.

Ericka Ann Lawson, Ph.D.

Visiting Professor
ealawson@swbell.net or elawson@rossu.edu

Reading: Meisenberg & Simmons: Principles of Medical Biochemistry,

4th edition, page 167-186.
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Learning Objectives

1. Describe the use of DNA microarrays for genetic diagnosis.

2. Explain the principle and interpret the results of genome-wide association
studies.
3. State the principle on which the Sanger sequencing method is based.
4. Provide a rough cost estimate for a genome sequence in 2018
5. Explain how next-generation sequencing is affecting medical science and medical
practice.
6. Describe the principle of array-based genomic hybridization, and the
situations in which it is used for diagnosis.
7. Distinguish between gene knock-in and knock-out, and describe the
methods used.
8. List the components needed for gene editing with the CRISPR-Cas9
system, and describe how the method works.
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DNA Microarrays (“DNA chips”)

This is the standard method when the aim is to genotype a large
number of DNA variants (SNPs, mutations) at the same time.
• Screening for presence of multiple SNPs or mutations in patients
• Ancestry testing, an important part of commercial genomics in
the US
• Genome-wide association studies (GWAS) for discovery of
medically important genetic variants
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DNA Microarrays: Procedure

1. A DNA chip (size of a microscope slide) is divided into up to a million

squares.
2. A different oligonucleotide (30-100 nt long) that is complementary
to an SNP allele is synthesized on each square.
3. Fluorescent-labelled restriction fragments of genomic DNA are
added.
4. Fluorescence is scanned and quantified automatically.

• The stringency of the washing steps of developing a micro array are very important as it was
in hybridization probes.
• The oligonucleotides are essentially ASOs (allele specific oligonucleotides).
• Each chip has up to 106 different ASOs
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Genome-Wide Association Studies (GWAS)

Purpose:
Discovery of polymorphisms that are statistically associated with a
disease or a quantitative trait.
Procedure:
- Make a microarray with probes for the alleles of many single-
nucleotide polymorphisms (SNPs) spread all over the genome.
- Add subject’s restriction-digested, fluorescence-labelled DNA.
- Scan the microarray, record genotypes of SNPs.
- Determine non-random associations with the studied trait.

• The patient DNA is cut with a restriction enzyme and then a DNA polymerase is used to fill in
the overhanging ends with NTPs where one or more have a fluorescent tag.
• The oligonucleotides in the array cover many different genes across all chromosomes.
• Rapid screening to determine which chromosome and which piece of the chromosome is
associated (may be near but does not have to be in the same gene) with the trait of interest.
Slide 6

SNPs and Educational

Attainment

• This research investigated the genetic correlation of SNP throughout the autosomal
chromosomes with Educational attainment.
• The dashed line is the level above which correlation is the possible relevant correlations.
• Higher significance of association suggests a stronger correlation.
Slide 7

Interpretation of GWAS Associations

Are the “GWAS hits” (SNPs that are statistically associated with a
phenotype) the gene variants that cause the observed outcome?
Most of the time, they are not.
The trait-associated SNPs merely flag a nearby correlated SNP. The
method is based on linkage disequilibrium between the tag SNP on
the DNA chip and the causal variant in the genome.
Therefore, all GWAS hits should be followed up by sequencing-based
studies.

• GWAS significance only suggests a correlation. Typically that is because the SNP probe may
identify genes that are genetically linked to the trait of interest.
• Linkage disequilibrium is the measure of how often two alleles co-segregate in genetics
relative to if they were sorted completely randomly.
• The identifying of correlated SNPs only tells the scientist where to begin looking more
closely. Additional work is required to sequence the nearby regions of the chromosomes
and them compare the “normal” to the “test” trait.
Slide 8

Normal cells Cancer cells

Study of gene
expression

Microarrays can be
used to compare
mRNA levels in
cancer cells with
mRNA levels in
normal cells.

• Similar to probing for association between genes or traits us DNA, RNA can be used to look
at RNA levels.
• mRNAs are copied into cDNAs using reverse transcriptase. Fluorescent nucleotides are
incorporated into the cDNAs
• Two types of DNA can be added at the same time and read simultaneously.
• This allows for the direct comparison of the 2 cDNAs expression.
• Spots that only have a single color show that only one of the cells has transcripts for
that gene and the other cell’s transcriptome is only present in miniscule amounts if
any for that gene.
• Intermediate colors indicate both cells have similar levels of the mRNA expressed.
Slide 9

DNA Sequencing
Importance: Identification of insertions, deletions and point mutations
that cannot be seen by other techniques.
What is sequenced? Mainly, restriction fragments or PCR products
generated from the DNA of interest.
For example, to find out whether there is a mutation in a gene:
- Amplify the exons with PCR
- Separate the PCR products by electrophoresis
- Sequence the PCR products
Exome sequencing or whole-genome sequencing is employed to
diagnose millions of mutations and polymorphisms at a time.

• Once a region of interest has been identified then the DNA in the regions is sequenced.
• This is the only way to tell the exact sequence of a gene.
• Products of the sequencing reaction are separated by size (usually on a Denaturing Gel
electrophoresis.
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The Sanger Method

This is the traditional method, used for the initial sequencing of the
human genome.
It is based on controlled interruption of DNA synthesis with a
dideoxyribonucleotide:
Slide 11

1) DNA Sequencing by the Sanger method is accomplished by using a trick of DNA replication.
2) dd means Dideoxy and means that the 3’ OH is replaced with a H on the ribose.
3) if there is not a 3’ hydroxyl on the nucleotide as the end of a nucleic acid polymer, the strand
cannot be further lengthened.
4) if the dideoxynucleosides are also fluorescently labelled then the DNA can be read by the
fluorometer of a DNA sequencing instrument.
Slide 12

Sanger Method:
Procedure
DNA and primer are mixed
with DNA polymerase and
2-deoxyribonucleotides. A
small amount of a dideoxy-
ribonucleotide is included,
causing chain termination.
The lengths of synthesized
products are determined by
electrophoresis.
Slide 13

Next-Generation Sequencing
Several new methods have been implemented in automatic DNA
sequencers. Increased efficiency by:
- Miniaturization
- Massively parallel sequencing
- Solid-phase techniques in many cases
- Computer programs aligning the sequences
Slide 14

Cost of Sequencing a Human Genome

Thanks to next-generation sequencing methods, the cost of a genome
sequence is now coming down to $1000 per genome. But: Actual cost
depends on utilization of machines, administrative overhead, CEO’s pay,
shareholders’ dividends etc.
Slide 15

Uses of Whole-Genome Sequencing

- Discovery tool for Mendelian (single-gene) disorders
- Discovery tool for rare variants contributing to polygenic diseases and
quantitative traits. Microarrays are sufficient for common variants.
- Diagnostic tool when genomic location of pathogenic mutation(s) is
uncertain
- Predictive testing for multiple disease susceptibilities
- Assessing genetic risks for future children
- Comprehensive embryo screening
- Cancer genomics, to find driver mutations in a patient’s cancer

• DNA sequencing may suggest a person’s susceptibility but is not the sole case for diagnosis.
• Insurance companies have attempted to gather whole genome data to use in computing the
insurability and rates for individuals at risk for contracting a disease.
Slide 16

Example: Hereditary Breast & Ovarian Cancer

This is a dominantly inherited condition that puts women at risk of

breast and ovarian cancer. 2 main genes:
BRCA1: 22 coding exons, > 5,500 bp
BRCA2: 26 coding exons, > 11,000 bp
Mutations in these genes account for 2-4% of breast cancer and ovarian
cancer, especially early-onset cancers.
Slide 17

BRCA Pedigree

• Genetic techniques are combined with biochemical DNA techniques to fine tune our
understanding of the inheritance of specific genes.
• Use of related individuals in the DNA sequencing allows for the reduction of non-important
differences to be eliminated quickly.
Slide 18

Sequencing Experience With BRCA1&2

Complete sequencing of both genes in >150,000 people

at Myriad Genetics alone:
• >10,000 mutations and benign or uncertain variants
identified
• Every week, 10-20 new missense variants were
detected that had never been seen before

The biggest challenge is to distinguish rare pathogenic

variants from rare harmless variants.
Slide 19

Detection of Copy Number Variations

This is done with array comparative genomic hybridization (aCGH).
1. A microarray is produced that has probes for monomorphic DNA
sequences scattered all over the genome.
2. A “normal” reference genome is cut up with a restriction enzyme, and
the fragments are labelled with green fluorescence.
3. The same is done with patient’s genome, but fragments are labelled
with red fluorescence.
4. The two DNA digests are mixed 1:1, and the red/green fluorescence
ratio determined for each microarray-bound fragment.
5. Reduced red/green ratio means deletion, increased red/green ratio
means duplication.

• This technique is similar in concept to other microarrays.

• The primary difference in this technique is the probes that are used for the microarray. This
technique only probes for genes that usually have only one copy per genome.
• This technique works well for small copy number variations but may fail as copy numbers
exceed an order of magnitude difference (tenfold).
• Is best for looking for gene duplication and gene deletions.
Slide 20

Log -ratio
Example:
6.66 Mb deletion of
Xp21 in a heterozygous
female. This deletion
causes glycerol kinase
deficiency, muscular
dystrophy and adrenal
hypoplasia in males.

• Patients with below normal copy variation are usually deletions.

• Patients with above normal copy variation are usually insertions.
Slide 21

Copy Number Variants

are common in all
genomes surveyed

•Blue = pathogenic
•Red = deletion
•Green = duplication
Slide 22

In cancer genomics,
array comparative
genomic hybridization
is used to detect
deletions and
amplifications in the
patient’s cancer.
Slide 23

A Case Study in the Value of Returning Incidental Findings

• HNPP is characterized by peripheral nerve pain, weakness, tingling or numbness. Often

there is a sense of pressure of the affected area. Episodes may last from a few minutes to
hours and days.
• Myocardial infarct has the overlapping symptom of radiating pain and sensation of pressure
Slide 24

Genome Editing
This is a set of techniques aimed at changing the genome of
a cell.
Uses for research:
- Knock-out mice – mice with a gene disrupted
- Knock-in or transgenic mice – mice with a gene modified
or inserted
Uses in medicine:
- Gene therapy, both for genetic and non-genetic diseases
- Germline gene modifications
Slide 25

How to Edit the Genome

Most techniques make use of the cell’s own DNA repair systems.
What do cells do
when there is a DNA
Non-homologous
double-strand break? end joining
Homologous
repair
They fix it!
1. Non-homologous
end joining: imprecise
2. Homologous
repair: requires
homologous DNA
Slide 26

Use of Sequence-Specific Nucleases

Aim: Repair or disrupt a gene
Strategy: Bring a “designer nuclease” into the cell that cleaves
the DNA at only one site. Then, let the cell repair the break.
Nucleases available:
- Zinc finger nucleases, (ZFN): Nuclease fused to sequence of
zinc fingers (the first technique developed)
- TALENs: Nuclease fused to a sequence of different targeting
modules (An improvement on ZFN)
- CRISPR-Cas9: Cas9 nuclease bound to a guide RNA that
base-pairs with the DNA (The method of choice today)
Slide 27

Example: Zinc finger nucleases. The FokI endonuclease is targeted to the

correct DNA sequence with a sequence of covalently fused zinc fingers.
Only the dimeric enzyme cleaves the DNA.

xxx

This method has the unfortunate side effect of integration of the targeted gene into place in the genome that are not
the same location as the parent gene. This has lead to the development of other diseases by patients many years
afterward. Thus, this method is rarely used since the introduction of CRISPR
Slide 28

Cas9 endonuclease is The CRISPR-Cas9 System

associated with a
guide RNA that base-
pairs with the DNA
target site. Two DNase
domains of Cas9 cut
the two DNA strands.
This system has been
developed from a
bacterial immune
system.
This is now the
standard system for
genome editing.
Slide 29

Genetically Modified Mice

For research purposes, mice can be genetically engineered:

- Knock out: A gene is disrupted (artificial loss-of-function
mutation)
- Knock in: A gene is changed into a different version, or a
foreign gene is introduced (transgenics). Can be used to create
gain-of-function mutations.
- Knock down: The genome is left unchanged, but a siRNA is
introduced that prevents translation of selected mRNAs.
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How to Make a Knock out Mouse or Transgenic Mouse

This is the straightforward method: Inject Cas9 with its guide RNA,
either with a donor template (for knock in) or without (for knock out).
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Another Way to Engineer a Mouse

The gene manipulation is done in cultured embryonic stem cells. The
engineered cells are injected into the embryo to make a chimeric
mouse.
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Uses of Knock out Mice

This procedure is used to determine the importance of genes:
Does homozygous loss of a gene cause abnormalities?
Does heterozygous loss of a gene cause abnormalities
(haploinsufficiency)?
What kind of disease can be expected when the corresponding
human gene is defective?

This has been done with most of our 19,000 genes.

Used to explore treatments for genetic diseases.

Slide 33

Knock-in Procedure
• Replace the normal mouse gene with a specifically changed version
• Example: Will the Δ-F508 mutation cause cystic fibrosis in mice?
If it does, we can try new treatments on this mouse.
Site of mutation
HR1 HR2
Insert
Mouse chromosome
Exons: 1 2 3 4

The same procedure can be used to repair defective genes in humans,

for example in zygotes of two parents with sickle cell disease.
Slide 34

Another Example: Amyotropic Lateral Sclerosis (ALS)

This is a deadly disease that destroys motor neurons in the spinal

cord.
• Mutation G93A in the gene for superoxide dismutase-1 (SOD1)
causes ALS in humans, most likely because the mutated
protein forms aggregates.
• Knock-out of SOD1 in mouse does not mimic ALS.
• Knock-in of G93A in the mouse does produce an ALS model.
Slide 35

Gene Therapy
This is the delivery of a foreign gene into a cell for therapy.
Examples:
- Get a functioning copy of a defective gene into cells of a patient
with a genetic disease (sickle cell, muscular dystrophy…)
- Get intact tumor suppressor genes into cancer cells
- Get anti-inflammatory genes into joints of arthritis patients
- Destroy DNA of an inserted retrovirus (AIDS!)

In many cases, these treatments are applied to cells from the

patient in vitro that then get re-inserted into the patient.
Slide 36

Gene Therapy: Challenges

1. How to get foreign DNA into the cell:
- Liposomes: not very effective.
- DNA viruses: cytotoxic, and gene expressed only temporarily.
- Retroviruses: insert themselves into genome.
2. How to get foreign DNA integrated into the genome: either with
retroviral vector or a designer nuclease (CRISPR-Cas9 etc).
3. Older methods caused insertion in random sites of the genome,
with bad side effects (cancer).

Generally, these methods are inefficient, with only a small

percentage of cells transformed.