Genetic Technologies paulj/tech_genetics.ppt

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?

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?

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

Sau3AI

GATCGATC CTAGCTAG

ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

Sau3AI

GATCGATC CTAGCTAG

Sau3AI

GATCGATC CTAGCTAG

ACTGTCGATGTCGTCGTCGTAGCTGCT GATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAG CATCGATCGA

ACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAG

GATCGTAGCTAGCT CATCGATCGA

ACTGTCGATGTCGTCGTCGTAGCTGCTGATGACAGCTACAGCAGCAGCATCGACGACT

TCGTAGCTAGCT AGCATCGATCGA

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

• DNA is negatively charged and will move through a gel matrix towards a positive electrode

• Shorter lengths move faster

Slow: 41 bpACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

Medium: 27 bpACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAG

Fast: 10 bpGATCGTAGCTAGCT CATCGATCGA

Recessive disease allele D is cut by Sma3AI:

Healthy H allele is not cut:

ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA

HH HD DD

A single strand of DNA will stick to its complement

Fragment length in bp

Southern blotting (named after Ed Southern)

DNA can copied by a polymerase enzyme

DNA polymerase

ACTGTCGATGTCGT

ACTGT ACTGTCGAT ACTGTCGATGT ACTGTCGATGTCGT ACTGTCGATGTCGTCGT ACTGTCGATGTCGTCGTCGT ACTGTCGATGTCGTCGTCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGAT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT

ACTGTCGATGTACTGTCGATGACTGTCGATACTGTCGAACTGTCGACTGTCACTGT

Fluorescence

TGTAGCT

T C G A T G T etc

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 templateHeat resistant DNA

polymeraseG, A, C, T

basesForward primer

Reverse primer

A thermal cycler (PCR machine)

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

le Theory

Practice

• 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…

• 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

• 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

A AA or AO

B BB or BO

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

OLA: oligonucleotide ligation assay Allele-specific oligonucleotide

Clin Biochem Rev (2006) 27: 63–75

No error 2% error

Common homozygote 960 940

Heterozygote 40 50

Rare homozygote 1 11

ARMS: amplification refractory mutation system

OLA: oligonucleotide ligation assay

Molecular beacon probes

Pyrosequencing

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

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

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)

• 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

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

Genetic Technologies paulj/tech_genetics.ppt

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