Identifying and Analysis of genes and Genetic Analysis of Gene Function

Question 1

How much does it cost to sequence your genome?

Answer 1

£7,800

Question 2

Give two ways we can find genes in the nucleotide sequence

Answer 2

Use gene prediction software

Use computer to translate the DNA in all 6 reading frames and then search for similarity proteins called blasts

Question 3

What is gene prediction software?

Answer 3

Where a sequence is scanned for promoters, start and stop code sequences and intron splicing sites
One cant be sure if this prediction is true

Question 4

Similarity between two proteins suggests what?

Answer 4

That proteins have evolved from the same common ancestor and they have similar molecular functions

Question 5

What are mircroarrays?

Answer 5

Allow us to compare the transcriptomes of different tissues to each other
High throughput, small scale, fast and automated

Question 6

What manufactures an array?

Answer 6

A very precise robot

Each position on the grid contains one cDNA and there is a spot for every gene in the genoeme

Question 7

What are the three different ways to identify genes?

Answer 7

1) By making a library of cDNA clones from mRNA
2) By making a library of genomic clones then make predictons based upon genomic sequence
3) Identify sets of interesting genes using microarray

Question 8

Give a way to analyse a gene

Answer 8

Reverse genetics eg in mice

Question 9

Describe gene replacement in a mouse

Answer 9

Usually makes small changes to the endogenous gene
eg mutations in humans could be tested for causing disease symptoms by making the same corresponding mutation in mice genes

Question 10

What is a knock out?

Answer 10

Where the gene is completetly removed to determine its function
To begin a knock out project you must first have a genomic clone of your gene

Question 11

How can we knockout a single gene in mice?

Answer 11

First acquire a genomic clone of the gene and insert NEO directly on to the econ (destroying the activity of the gene)
TK is placed off to one side

Question 12

Why are the sequences from the target genes called homologous arms?

Answer 12

Because they are the only sequence with homology to the mouse genome

Question 13

The DNA contrsuct is inserted in to what?

Answer 13

Embryonic stem cells

Question 14

Is the process of recombining the construct in to the mouse genome efficient?

Answer 14

No

Question 15

When homologous recombination does occur, which gene is lost?

Answer 15

TK

Question 16

How are knock outs identified?

Answer 16

Double selection

Question 17

How are cells that are a result of homologous recombination selected for?

Answer 17

Cells that have integrated NEO are now able to grow on a neomycin media
Cells that have integrated TK and Neo wil die when grown on GANC media

Question 18

How is a single gene targeted in mice?

Answer 18

1) The selected cell line is reintroduced in to the mice embryos
2) The first generation is mosaic and so are their gonads
3) These mosaic animals are then bred to generate non-mosaic carriers of the transgene
4) The carriers are then interbred to create homozygous mutant mice

Question 19

What is one in forward genetics?

Answer 19

1) Randomly mutate the genome
2) Look for interesting phenotypes in the offspring
3) Identify the gene that causes the defect

Question 20

Which are the two main model organisms used in forward genetic screens?

Answer 20

Drosophila and zebrafish

Question 21

What is reverse genetics?

Answer 21

Where we already know the gene and we want to find its function

Question 22

Describe the steps in a genetic fly screen

Answer 22

1) Mutagenise male: each serm has a different set of mutations by EMS - this male is heterozygous for the mutation
2) Outcross male to wildtype female
3) Incross the family to see what homozygour embryos look like
4) 1/4 of the offspring will show the phenotype if there is one

Question 23

How many generations does it take to make a mutation homozygous?

Answer 23

3

Question 24

How do you test if mutations are on different alleles if two mutations show the same phenotype?

Answer 24

Complementation testing

Question 25

What is the result of a complementation test if the mutation are alleles of the same gene?

Answer 25

M1/+ x M2/+
= M1/M2
= 1/4 mutant offspring
Mutations fail to complement so they are alleles of the same gene

Question 26

What is the result of a complementation test if mutations are on different genes?

Answer 26

M1/+x M3/+
= M1+ and M3/+
No offspring with the phenotype
This means they complement so they are on different genes

Question 27

What is linkage analysis?

Answer 27

Analyses recombination between an allele and a marker on the same chromosome
This is a way of determining if an allele and a marker are linked by looking at the frequency of cross over in meiosos

Question 28

Offspring that have which chromosomes are the product of recombination events?

Answer 28

aB or Ab

Question 29

How is recombinant frequency calculated?

Answer 29

Counting the number of recombinant gametes (R)/total number of gametes (T)

Question 30

How do you calculate the centimorgan?

Answer 30

R/T x 100 = cM

cM is a measurement of genetic distance

Question 31

What are SNPs?

Answer 31

Single nucleotide polyorphisms
They are markers that vary from individual to individual
There are over 1 million placed in the human genome

Question 32

How can we know if a gene is linked to an SNP?

Answer 32

DNA samples are taken from a parent and a child with a disease
If an SNP is always present in diseased children but not in healthy ones, then we know the gene is linked to the SNP
If an SNP is closed to a gene then this gene is a candiate gene

Question 33

How can mutations affect gene function?

Answer 33

1) Changes in regulatory sequences - affects transcription
2) Changes in non-coding sequences - may affect splicing, stability or translation
3) Changes in coding sequence - may alter amino acids affecting the folding of the protein or truncation

Question 34

What happens in an amorphic mutation?

Answer 34

Missense mutation that completely inactivates the protein if -/-
If +/- there is enough gene product = haplosuffieciency

Question 35

What happens in a hyomorphic mutation?

Answer 35

In +/- there is enough gene product but on -/- the protein only completes half of its function

Question 36

What happens in an antimorphic mutation?

Answer 36

+/- the mutant form poisons the WT protein