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Genetic Technologies
http://www.stats.gla.ac.uk/~paulj/tech_genetics.ppt
Overview
• Why learn about genetic technologies?• The molecular geneticist’s toolkit• Genetic markers• Microarray assays• Telomeres• RNA interference (RNAi)
Why learn about genetic technologies?
Why learn about genetic technologies?
We need to understand the processes that generated the data
• Understanding of biology obviously necessary• Understanding of lab techniques will enhance our ability to
assess data reliability• Errors in any measurement can lead to loss of power or
bias• Some genetic analyses are particularly sensitive to error
because– they depend on the level of identity between DNA
sequences shared by relatives– the more data is collected, the greater the chance of
false differences
Why learn about genetic technologies?
A
B
Genotype
177, 179
179, 179
Individual
• What is the probability that the observed genotype is wrong?
• Is this probability the same for all observed genotypes?
• What impact will a realistic range of errors have on power?
The molecular geneticist’s toolkit
Most genetic technologies are based on four properties of DNA
1. DNA can be cut at specific sites (motifs) by restriction enzymes
2. Different lengths of DNA can be size-separated by gel electrophoresis
3. A single strand of DNA will stick to its complement (hybridisation)
4. DNA can copied by a polymerase enzyme• DNA sequencing• Polymerase chain reaction (PCR)
• Restriction enzymes cut double-stranded DNA at specific sequences (motifs)
• E.g. the enzyme Sau3AI cuts at the sequence GATC• Most recognition sites are palindromes: e.g. the reverse
complement of GATC is GATC• Restriction enzymes evolved as defence against foreign
DNA
DNA can be cut at specific sites (motifs)by an enzyme
Sau3AI
GATCGATC CTAGCTAG
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can be cut at specific sites (motifs)by an enzyme
Sau3AI
GATCGATC CTAGCTAG
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can be cut at specific sites (motifs)by an enzyme
Sau3AI
GATCGATC CTAGCTAG
ACTGTCGATGTCGTCGTCGTAGCTGCT GATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAG CATCGATCGA
DNA can be cut at specific sites (motifs)by an enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAG
GATCGTAGCTAGCT CATCGATCGA
ACTGTCGATGTCGTCGTCGTAGCTGCTGATGACAGCTACAGCAGCAGCATCGACGACT
TCGTAGCTAGCT AGCATCGATCGA
DNA can be cut at specific sites (motifs)by an enzyme
Different lengths of DNA can be separated by gel electrophoresis
• DNA is negatively charged and will move through a gel matrix towards a positive electrode
• Shorter lengths move faster
Different lengths of DNA can be separated by gel electrophoresis
• DNA is negatively charged and will move through a gel matrix towards a positive electrode
• Shorter lengths move faster
Different lengths of DNA can be separated by gel electrophoresis
Slow: 41 bpACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
Medium: 27 bpACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAG
Fast: 10 bpGATCGTAGCTAGCT CATCGATCGA
F
M
S
Different lengths of DNA can be separated by gel electrophoresis
Recessive disease allele D is cut by Sma3AI:
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
Healthy H allele is not cut:
ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA
F
M
S
HH HD DD
Different lengths of DNA can be separated by gel electrophoresis
F
M
S
HH HD DD
A single strand of DNA will stick to its complement
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
60°C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
95°C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
60°C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
Fragment length in bp
Fra
gmen
t fr
eque
ncy
Flo
ures
cenc
e
A single strand of DNA will stick to its complement
A single strand of DNA will stick to its complement
Southern blotting (named after Ed Southern)
A single strand of DNA will stick to its complement
Southern blotting (named after Ed Southern)
A single strand of DNA will stick to its complement
A single strand of DNA will stick to its complement
A single strand of DNA will stick to its complement
DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA polymerase
C
CC
CCC
G
G
G
G
G
GG
G
G
T T
T
T
A
T
T
A
A
A
AA
A
A
A
DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA polymerase
C
CC
CCC
G
G
G
G
G
GG
G
G
T T
T
T
A
T
T
A
A
A
AA
A
A
A
DNA can copied by a polymerase enzyme
ACTGTCGATGTCGT
DNA can copied by a polymerase enzyme
ACTGT ACTGTCGAT ACTGTCGATGT ACTGTCGATGTCGT ACTGTCGATGTCGTCGT ACTGTCGATGTCGTCGTCGT ACTGTCGATGTCGTCGTCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGAT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
DNA can copied by a polymerase enzyme
ACTGTCGATGTACTGTCGATGACTGTCGATACTGTCGAACTGTCGACTGTCACTGT
Fluorescence
Tim
e
Flu
ores
cenc
e
Time
TGTAGCT
T C G A T G T etc
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
Polymerase chain reaction (PCR)• A method for producing large (and therefore analysable) quantities of a specific
region of DNA from tiny quantities• PCR works by doubling the quantity of the target sequence through repeated cycles
of separation and synthesis of DNA strands
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
A
C
T
G
DNA templateHeat resistant DNA
polymeraseG, A, C, T
basesForward primer
Reverse primer
A thermal cycler (PCR machine)
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
Increase in DNA quantity in PCR
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
1.0E+10
1.0E+11
0 5 10 15 20 25 30 35Cycle number
Qu
an
tity
of
DN
A r
ela
tiv
e t
o in
itia
l sa
mp
le Theory
Practice
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
• PCR can generate 100 billion copies from a single DNA molecule in an afternoon• PCR is easy to execute• The DNA sample can be pure, or it can be a minute part of an extremely complex
mixture of biological materials• The DNA may come from
– a hospital tissue specimen– a single human hair– a drop of dried blood at the scene of a crime– the tissues of a mummified brain– a 40,000-year-old wooly mammoth frozen in a glacier.
In the words of its inventor, Kary Mullis…
DNA can copied by a polymerase enzyme
The molecular geneticist’s toolkit
• Specific DNA-cutting restriction enzymes
• DNA size separation by gel electrophoresis
• Hybridisation using labelled DNA probes
• Synthesis of DNA using DNA polymerase (PCR)
Genetic markers
Genetic markers
• What are they?– Variable sites in the genome
• What are their uses?– Finding disease genes– Testing/estimating relationships – Studying population differences
Eye colour
Phenotype Genotype
Brown eyes BB or Bb
Blue eyes bb
ABO blood group
Phenotype Genotype
AB AB
A AA or AO
B BB or BO
O OO
The ideal genetic marker
• Codominant• High diversity• Frequent across whole genome• Easy to find• Easy to assay
Modern genetic markers: SNPs
• SNPs are single nucleotide polymorphisms
• Usually biallelic, and one allele is usually rare
• Can be protein-coding or not
• This example is a T/G SNP. An individual can be TT, TG, GG
Healthy allele A is cut by Sma3AI:ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
Recessive disease B allele is not cut:ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA
Modern genetic markers: SNPs
OLA: oligonucleotide ligation assay Allele-specific oligonucleotide
Clin Biochem Rev (2006) 27: 63–75
Modern genetic markers: SNPs
Modern genetic markers: SNPs
No error 2% error
Common homozygote 960 940
Heterozygote 40 50
Rare homozygote 1 11
Modern genetic markers: SNPs
Clin Biochem Rev (2006) 27: 63–75
Modern genetic markers: SNPs
ARMS: amplification refractory mutation system
Clin Biochem Rev (2006) 27: 63–75
Modern genetic markers: SNPs
OLA: oligonucleotide ligation assay
Clin Biochem Rev (2006) 27: 63–75
Modern genetic markers: SNPs
Molecular beacon probes
Clin Biochem Rev (2006) 27: 63–75
Modern genetic markers: SNPs
Pyrosequencing
Clin Biochem Rev (2006) 27: 63–75
Modern genetic markers: microsatellites
• Microsatellites are short tandem repeats (STR, also SSR)
• Usually high diversity
• Usually not in protein coding sequence
• This example is an (AC)n repeat; a genotype is usually written n,n
• With k alleles there are k(k+1)/2 possible unordered genotypes
ACTGTCGACACACACACACACGCTAGCT (AC)7
TGACAGCTGTGTGTGTGTGTGCGATCGA
ACTGTCGACACACACACACACACGCTAGCT (AC)8
TGACAGCTGTGTGTGTGTGTGTGCGATCGA
ACTGTCGACACACACACACACACACACGCTAGCT (AC)10
TGACAGCTGTGTGTGTGTGTGTGTGTGCGATCGA
ACTGTCGACACACACACACACACACACACACGCTAGCT (AC)12
TGACAGCTGTGTGTGTGTGTGTGTGTGTGTGCGATCGA
7 8 9 12
7 7,7
8 7,8 8,8
9 7,9 8,9 9,9
12 7,12 8,12 9,12 12,12
Modern genetic markers: microsatellites
Modern genetic markers: microsatellites
Modern genetic markers: microsatellites
Modern genetic markers: microsatellites
Microsatellites versus SNPs
Microsatellites SNPs
Codominant Yes Yes
Diversity High Low
Frequent 10,000s 3 million
Easy to assay Yes Yes
Easy to find No No, but…
Uses of SNPs and microsatellites
• SNPs– The HapMap project has discovered millions of SNPs– Their high density in the genome makes them ideal for
association studies, where markers very close to disease genes are required
• Microsatellites– More suitable for family-based studies, where high variation is
valuable and lower levels of resolution are required
Overview
• Why learn about genetic technologies?• The molecular geneticist’s toolkit• Genetic markers• Microarrays• Telomeres• RNA interference (RNAi)
The molecular geneticist’s toolkit
• Specific DNA-cutting restriction enzymes
• DNA size separation by gel electrophoresis
• Hybridisation using labelled DNA probes
• Synthesis of DNA using DNA polymerase (PCR)
Overview
• Why learn about genetic technologies?• The molecular geneticist’s toolkit• Genetic markers• Microarrays• Telomeres• RNA interference (RNAi)
Microarrays
Gene expression
• Transcription: – DNA gene → mRNA– in nucleus
• Translation: – mRNA → protein– in cytoplasm
• Microarrays use mRNA as a marker of gene expression
Nucleus Cytoplasm
What are microarrays?
• A microarray is a DNA “chip” which holds 1000s of different DNA sequences
• Each DNA sequence might represent a different gene• Microarrays are useful for measuring differences in gene
expression between two cell types• They can also be used to study chromosomal
aberrations in cancer cells
Principles behind microarray analysis
• Almost every body cell contains all ~25,000 genes• Only a fraction is switched on (expressed) at any time in
any cell type• Gene expression involves the production of specific
messenger RNA (mRNA)• Presence and quantity of mRNA can be detected by
hybridisation to known RNA (or DNA) sequences
What can microarray analysis tell us?
• Which genes are involved in– disease?– drug response?
• Which genes are – switched off/underexpressed?– switched on/overexpressed?
Before microarrays: northern blotting
• Extract all the mRNA from a cell• Size-separate it through a gel• Measure level of expression using a probe made from your gene
of interest
Northern blotting: still useful for single-gene studies
Microarray analysis: probe preparation
Microarray analysis: target preparation
Arthritis Research & Therapy 2006, 8:R100
Microarrays can be used to diagnose and stage tumours, and to find genes involved in tumorigenesis
• Copy number changes are common in tumours• Loss or duplication of a gene can be a critical stage in tumour
development
Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 202122
BMC Cancer 2006, 6:96
Problems of microarray analysis
• Gene expression ≠ mRNA concentration• Easy to do, difficult to interpret• Standardisation between labs• Lots of noise, lots of genes (parameters)
– e.g. p = 10,000 • low sample size
– e.g. n = 3
Telomeres
Telomeres and telomerase
• Telomeres are repetitive DNA sequences at the ends of chromosomes
• They protect the ends of the chromosome from DNA repair mechanisms
• In somatic cells they shorten at every cell division, leading to aging
• In germ cells they are re-synthesised by the enzyme telomerase
Telomere
Centromere
Telomere
Why do we need telomeres?
• At every cell division each chromosome must be replicated
• DNA is synthesised in one direction only
• The “lagging strand” is synthesised “backwards” in 100–200 bp chunks
Leading strand
• This isn’t a problem for the leading strand…
Lagging strand
• …but 100–200 bp of single stranded DNA are left hanging at the end of the lagging strand, and are lost.
Terminal (GGGTTA)n repeats buffer DNA loss
In germ cells, telomerase “rebuilds” telomeres
Health implications of telomere shortening: aging
Health implications of telomere shortening: cancer
• Cancer tumour cells divide excessively, and will die unless they activate telomerase
• Telomerase activation is an important step in many cancer cell types
• Telomere length can be used to diagnose tumours
• Telomerase is a potential target of cancer therapy
Measuring telomeres
• Two principal methods
Southern blotting Quantitative PCR (qPCR)
Measuring telomeres
RNA interference (RNAi)
What is RNAi?
• Generally genes are studied through the effects of knockout mutations in particular experimental organisms
• RNAi is a quick and easy technique for reducing gene function without the necessity of generating mutants that can be applied to any organism
• It has the potential to treat diseases caused by over-expression of genes
Principles of RNA interference (RNAi)
• Injection of double-stranded RNA (dsRNA) complementary to a gene silences gene expression by – destruction of mRNA– transcriptional silencing– stopping protein synthesis
• Gene expression can be switched off in specific tissues or cells by the injection of specific dsRNA
RNAi
http://www.nature.com/focus/rnai/animations/index.html
Uses of RNAi
• Investigating role of genes by knocking down (not out) gene expression in specific tissues at specific developmental stages
• Potential use in gene therapy– macular degeneration: two phase I trials currently under way– therapies being developed for HIV, hepatitis, cancers
Limitations of RNAi
• Target specificity: how do you know the dsRNA isn’t interfering with other genes?– Interpretation of results– Risks for gene therapy
• Function isn’t knocked out, it’s reduced– Knockdown may not reveal gene function– Might not give therapeutic effect