4. Analysing genes IIIsolate mutants*

classify mutants by complementation analysis

study phenotype of mutants

use mutant to isolate gene

sequence gene

analyse with bioinformatics (eg similarities to other genes?)

express gene product & study function

1

.

Ade wild type

Ade-x

x

Ade

mutant 1

Using the mutant to isolate the gene

x

Ade

put individual genes into Ade- cell

2

3

Plasmid definition!

“small (5-100 kb) circular DNA molecule that replicates in bacterial cells independently of bacterial chromosome”!!

- contain antibiotic resistance genes !

- can be modified so they replicate in yeast as well as bacteria

3

identify plasmid which complements Ade- phenotype .

Ade wild type

Ade-x

x

Ademutant 1

Using the mutant to isolate the gene

4

Restriction enzymes• Restriction enzymes are

bacterial endonucleases that cut specific DNA sequences

• Many (type II) cut within or near the specific sequence

• Recognition sequence usually 4-8 bp and often palindromic

• Incisions often staggered giving rise to ‘sticky ends’

• Evolved as bacteriophage defence

5’ GAATTC 3’ !3’ CTTAAG 5’

EcoRI cuts on average

1 in 46

5

5'CATGACGTCAT3'GTACTGCAGTA

GCTTAAAATTC G

ACGTAGCTGATC 3'TGCATCGACTAG 5'

Eco RI

AATTC G

ACGTAGCTGATC 3'TGCATCGACTAG 5'

5'CATGACGTCAT3'GTACTGCAGTA

GCTTAA

5'CATGACGTCAT3'GTACTGCAGTA

GCTTAAAATTC G

ACGTAGCTGATC 3'TGCATCGACTAG 5'

OH

OH

reduce temperature

H-bonding between sticky ends allows cut fragments to associate at low temperatures

6

DNA ligase• DNA ligase joins 5’ phosphate & 3’OH of

adjacent nucleotides - thus sealing nicks in DNA strand

• ligases are found in all organisms but phage T4 ligase is used for in vitro recombination

7

Making a gene library wild type yeast DNAplasmid

cleavage and ligation

Each colony = plasmid clone, containing a different yeast genome fragment

transformation into E.coli

with enough colonies recombinant plasmids all yeast sequences will be represented

8

ADE1

9

10

Transform gene library into yeast ade1 mutant

yeast gene!on !plasmid CDC2 RAD1 POL2 ADE1

growth on

medium lacking adenine

X X X

xade1

10

x

wt ADE1

mutant ade1

prepare total DNA

transform bacteria selecting for marker on

plasmidprepare plasmid DNA (microgram

quantities)

Final steps in ADE1 gene purification

11

How can the function of individual genes be understood?Isolate mutants*

classify mutants by complementation analysis

study phenotype of mutants

use mutant to isolate gene

sequence gene

analyse with bioinformatics (eg similarities to other genes?)

express gene product & study function

12

lllllllllll

we need •DNA polymerase (Taq) •Template DNA •DNA primer •dNTPs and fluorescently-labelled dideoxyNTPs

Dideoxy (Sanger) sequencing

denature by

heating to 95°C

13

Dideoxy (Sanger) sequencing

http://www.dnai.org/b/index.html14

media.invitrogen.com.edgesuite.net/ab/applications-technologies/pharma-biotherapeutics/DNA_sequencing.swf

Polymerase extends primer from a defined start !When a fluorescent dideoxy is incorporated, synthesis is terminated

15

16

Millions of fragments are synthesised, terminating in the same fluorescent nucleotide

16

17

After the synthesis reaction the products are separated by capillary electrophoresis - smaller fragments migrate faster. After separation the fragments are scanned by a laser and the colour of the light emitted indicates the terminating nucleotide.

17

18

Output from the Zoology ABI sequencer

18

How are the data analysed?

Only about 500-1000 bp can be sequenced in one reaction Sequences from different primers are assembled by computer to highlight errors and generate the complete sequence of the gene Assembly of raw sequence reads is called a contig

19 20

!ATGTCAATTACGAAGACTGAACTGGACGGTATATTGCCATTGGTGGCCAGAGGTAAAGTTAGAGACATATATGAGGTAGACGCTGGTACGTTGCT

• identify where gene is in sequence, promoter, coding region etc

• predict amino acid sequence of product

• compare to database of other sequences to see if similar to previously characterised genes

express gene product & study function

ADE1

How are the data analysed?

analyse with bioinformatics

21

How can the function of individual genes be understood?

strategy I !

mutant !

!

!

gene

strategy 2 !

genome !

!

!

gene

22

How can the function of individual genes be understood?sequence genome

identify genes by bioinformatics

predict likely function of genes from bioinformatics

purify genes of interest by PCR

mutate gene in vivo to determine phenotype

express gene product & study function

23

Parallel sequencing methods allow rapid genome sequencing

e.g. illumina (based on dideoxy method)

<£1 for 106 bases human genome - 1 day

24

http://www.wellcome.ac.uk/Education-resources/Education-and-learning/Resources/Animation/WTX056051.htm

Illumina sequencing method

25

Bioinformatic analysis of sequence data allows identification of genes

segment of fission yeast genomehttp://www.pombase.org

26

http://dx.doi.org/10.6084/m9.figshare.722952

27

Summary - Gene identification

strategy I !

mutant !

!

!

gene

strategy 2 !

genome !

!

!

gene

!• mutant phenotype indicates gene

function Disadvantages

Advantages

• all genes potentially identifiable

• expensive, but increasingly less so • may not identify gene function • small genes may be difficult to

detect

!• requires genetically amenable

organism • piecemeal, one mutant screen

identifies a handful of genes

28

Questions you should be able to answer from this lecture !

- show in diagrams how dideoxy sequencing works - what is a contig? - explain how genes can be identified without using mutants - what features of restriction enzymes make them useful for in vitro recombination? - what is a plasmid and how does it differ from a chromosome?

29