Genetic Technologies

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Genetic Technologies
http//www.stats.gla.ac.uk/paulj/tech_genetics.pp
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Overview

• Why learn about genetic technologies?
• The molecular geneticists toolkit
• Genetic markers
• Microarray assays
• Telomeres
• RNA interference (RNAi)

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Why learn about genetic technologies?
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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

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Why learn about genetic technologies?
Genotype
Individual
A
177, 179
B
179, 179

• 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?

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The molecular geneticists toolkit
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Most genetic technologies are based on four
properties of DNA

• DNA can be cut at specific sites (motifs) by
  restriction enzymes
• Different lengths of DNA can be size-separated by
  gel electrophoresis
• A single strand of DNA will stick to its
  complement (hybridisation)
• DNA can copied by a polymerase enzyme
• DNA sequencing
• Polymerase chain reaction (PCR)

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DNA can be cut at specific sites (motifs)by an
enzyme

• 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

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DNA can be cut at specific sites (motifs)by an
enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGA
CAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
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DNA can be cut at specific sites (motifs)by an
enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGA
CAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
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DNA can be cut at specific sites (motifs)by an
enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCT
GATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAG
CATCGATCGA
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DNA can be cut at specific sites (motifs)by an
enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCT TGACAGCTACAGCAGCAGCAT
CGACGACTAG GATCGTAGCTAGCT CATCGATCGA ACTGTCGA
TGTCGTCGTCGTAGCTGCTGA TGACAGCTACAGCAGCAGCATCGACGAC
T TCGTAGCTAGCT AGCATCGATCGA
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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

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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

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Different lengths of DNA can be separated by gel
electrophoresis
Slow 41 bp ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTA
GCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA Med
ium 27 bp ACTGTCGATGTCGTCGTCGTAGCTGCT TGACAGCTACA
GCAGCAGCATCGACGACTAG Fast 10
bp GATCGTAGCTAGCT CATCGATCGA
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Different lengths of DNA can be separated by gel
electrophoresis
Recessive disease allele D is cut by
Sma3AI ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA Healthy
H allele is not cut ACTGTCGATGTCGTCGTCGTAGCTGCTG
AGCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTCGCATCG
ATCGA
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Different lengths of DNA can be separated by gel
electrophoresis
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A single strand of DNA will stick to its
complement
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
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A single strand of DNA will stick to its
complement
60C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
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A single strand of DNA will stick to its
complement
95C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
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A single strand of DNA will stick to its
complement
60C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
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A single strand of DNA will stick to its
complement
Fragment frequency
Flourescence
Fragment length in bp
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A single strand of DNA will stick to its
complement
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A single strand of DNA will stick to its
complement
Southern blotting (named after Ed Southern)
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A single strand of DNA will stick to its
complement
Southern blotting (named after Ed Southern)
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A single strand of DNA will stick to its
complement
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A single strand of DNA will stick to its
complement
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A single strand of DNA will stick to its
complement
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DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGA
CAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
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DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGA
CAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A
G
T
G
C
A
G
A
C
T
G
G
A
A
G
A
G
T
T
C
T
C
C
C
G
A
T
G
A
A
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DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGA
CAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A
G
T
G
C
A
G
A
C
T
G
G
A
A
G
A
G
T
T
C
T
C
C
C
G
A
T
G
A
A
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DNA can copied by a polymerase enzyme
ACTGTCGATGTCGT
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DNA can copied by a polymerase enzyme
ACTGT ACTGTCGAT ACTGTCGATGT
ACTGTCGATGTCGT ACTGTCGATGTCGTCGT
ACTGTCGATGTCGTCGTCGT ACTGTCGATGTCGTCGTCGTAGCT
ACTGTCGATGTCGTCGTCGTAGCTGCT ACTGTCGATGTCGTCGTCGT
AGCTGCTGAT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGT
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCT
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
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DNA can copied by a polymerase enzyme
ACTGTCGATGT ACTGTCGATG ACTGTCGAT ACTGTCGA ACTGTC
G ACTGTC ACTGT
T G T A G C T
Time
Fluorescence
T C G A T G T etc
Fluorescence
Time
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DNA can copied by a polymerase enzyme
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DNA can copied by a polymerase enzyme
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DNA can copied by a polymerase enzyme
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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

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DNA can copied by a polymerase enzyme
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DNA can copied by a polymerase enzyme
G
A
C
T
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DNA can copied by a polymerase enzyme
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DNA can copied by a polymerase enzyme
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DNA can copied by a polymerase enzyme
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DNA can copied by a polymerase enzyme
In the words of its inventor, Kary Mullis

• 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.

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DNA can copied by a polymerase enzyme
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The molecular geneticists toolkit

• Specific DNA-cutting restriction enzymes
• DNA size separation by gel electrophoresis
• Hybridisation using labelled DNA probes
• Synthesis of DNA using DNA polymerase (PCR)

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Genetic markers
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Genetic markers

• What are they?
• Variable sites in the genome
• What are their uses?
• Finding disease genes
• Testing/estimating relationships
• Studying population differences

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Eye colour
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ABO blood group
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The ideal genetic marker

• Codominant
• High diversity
• Frequent across whole genome
• Easy to find
• Easy to assay

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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 ACTGTCGATGTCGTC
GTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCG
ACGACTAGCATCGATCGA Recessive disease B allele is
not cut ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA
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Modern genetic markers SNPs
Allele-specific oligonucleotide
OLA oligonucleotide ligation assay
Clin Biochem Rev (2006) 27 6375
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Modern genetic markers SNPs
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Modern genetic markers SNPs
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Modern genetic markers SNPs
Clin Biochem Rev (2006) 27 6375
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Modern genetic markers SNPs
ARMS amplification refractory mutation system
Clin Biochem Rev (2006) 27 6375
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Modern genetic markers SNPs
OLA oligonucleotide ligation assay
Clin Biochem Rev (2006) 27 6375
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Modern genetic markers SNPs
Molecular beacon probes
Clin Biochem Rev (2006) 27 6375
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Modern genetic markers SNPs
Pyrosequencing
Clin Biochem Rev (2006) 27 6375
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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(k1)/2 possible
  unordered genotypes

ACTGTCGACACACACACACACGCTAGCT (AC)7 TGACAGCTGTGTGT
GTGTGTGCGATCGA ACTGTCGACACACACACACACACGCTAGCT (A
C)8 TGACAGCTGTGTGTGTGTGTGTGCGATCGA ACTGTCGACACACA
CACACACACACACGCTAGCT (AC)10 TGACAGCTGTGTGTGTGTGTGT
GTGTGCGATCGA ACTGTCGACACACACACACACACACACACACGCTAG
CT (AC)12 TGACAGCTGTGTGTGTGTGTGTGTGTGTGTGCGATCGA
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Modern genetic markers microsatellites
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Modern genetic markers microsatellites
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Modern genetic markers microsatellites
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Modern genetic markers microsatellites
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Microsatellites versus SNPs
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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

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Overview

• Why learn about genetic technologies?
• The molecular geneticists toolkit
• Genetic markers
• Microarrays
• Telomeres
• RNA interference (RNAi)

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The molecular geneticists toolkit

• Specific DNA-cutting restriction enzymes
• DNA size separation by gel electrophoresis
• Hybridisation using labelled DNA probes
• Synthesis of DNA using DNA polymerase (PCR)

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Overview

• Why learn about genetic technologies?
• The molecular geneticists toolkit
• Genetic markers
• Microarrays
• Telomeres
• RNA interference (RNAi)

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Microarrays
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Gene expression

• Transcription
• DNA gene ? mRNA
• in nucleus
• Translation
• mRNA ? protein
• in cytoplasm
• Microarrays use mRNA as a marker of gene
  expression

Nucleus
Cytoplasm
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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

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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

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What can microarray analysis tell us?

• Which genes are involved in
• disease?
• drug response?
• Which genes are
• switched off/underexpressed?
• switched on/overexpressed?

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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

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Northern blotting still useful for single-gene
studies
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Microarray analysis probe preparation
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Microarray analysis target preparation
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Arthritis Research Therapy 2006, 8R100
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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, 696
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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

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Telomeres
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Telomeres and telomerase
Telomere

• 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

Centromere
Telomere
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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 100200 bp chunks

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Leading strand

• This isnt a problem for the leading strand

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Lagging strand

• but 100200 bp of single stranded DNA are left
  hanging at the end of the lagging strand, and are
  lost.

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Terminal (GGGTTA)n repeats buffer DNA loss
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In germ cells, telomerase rebuilds telomeres
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Health implications of telomere shortening aging
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(No Transcript)
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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

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Measuring telomeres

• Two principal methods
• Southern blotting Quantitative PCR (qPCR)

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Measuring telomeres
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RNA interference (RNAi)
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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

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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

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RNAi
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http//www.nature.com/focus/rnai/animations/index.
html
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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

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Limitations of RNAi

• Target specificity how do you know the dsRNA
  isnt interfering with other genes?
• Interpretation of results
• Risks for gene therapy
• Function isnt knocked out, its reduced
• Knockdown may not reveal gene function
• Might not give therapeutic effect