24. Making and screening genomic DNA

Question 1

why are genomic libaries used instead of PCR?

Answer 1

because when cloning a gene using a PCR, a primer must be made that is complimetry to the DNA - there the DNA sequence must be known.

If the DNA sequence is not know, can use a genomic library

Question 2

define genomic libaries

Answer 2

A set of cloned DNA fragments that together represent the total genome of a particular organism.

The DNA is stored in a population of identical vectors, each containing a different insert of DNA.

Question 3

define cDNA libararies

Answer 3

a set of cloned DNA fragements that together represent the mRNA from a particular organism or tissue

Question 4

whats the difference between genomic and cDNA libraries?

Answer 4

genomic libareis look at the genome of an organism

cDNA look at the mRNA of an organism

Question 5

what are the four steps to make a library?

Answer 5

1. start with millions of copies of the gene
2. fragment the DNA - makes it manageable
3. clone it
4. find the gene of interest

Question 6

what is a DNA probe? what is it used for?

Answer 6

a single stranded segment that is complimentary to a gene of DNA.

Its used to find the presence of that specific complementary sequence in a genomic library

Question 7

where are two ways probes can be obtained

Answer 7

1. previously cloned genes (related genes or organisms)
2. derived from a known protein sequence

Question 8

what are three steps to design a probe from a known amino acid sequence

Answer 8

1. select amino acid sequence with minimal degrenacy
2. determine all posisble nucleotide sequences that could end as that amino acid
3. order a set of degerate oligonucleotides matching the DNA sequence

Question 9

how can hybridisation conditions be altered to change the amount of mismatches that occur? 3 points

Answer 9

1. increase annealing temperature
2. reduce salt concentrations
3. increase detergent concentrations