Genetics - Restriction Enzymes/Cloning

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What is a restriction enzyme?

they are endonucleases that exist naturally in prokaryotes. They recognize foreign DNA sequences and cut them into little pieces for further degradation by host exonucleases
they RESTRICT what type of DNA can exist in the host

what is the purpose of restrictive enzymes in bacteria?

they are the bacterias defense, as bacteria have no immune system

What do endonucleases allow us to do?

allows cutting of the DNA in predictable places exactly

What does Type 2 REN do?

recognize inverted palindromic sequences in DNA
- each REN has a unique recognition sequence
-when cutting occurs it will either leave cohesive or blunt ends

What does electrophroesis do?

distinguishes DNA fragments according to size

Define electrophoresis

the movement of charged in an electric field . DNA in solution is weakly acidic (-ve charge)
-the larger the DNA molecule, the slower it moves through the gel matrix

What is a restriction map?

shows the order of different size fragments within a clone. consist of linear (or circular) diagrams of the sites along a DNA segment at which one or another restriction enzyme cleaves the molecule

what is molecular cloning?

The process of using living cells to make many exact replicas of a fragment of foreign DNA

how does molecular cloning work?

1. take a DNA fragment and insert the fragment into a specialized chromosome like carrier called a VECTOR.

2. transfer the vector with its insert into cells. copies will be made throught replication. these are therefore known as DNA clones.

What is the reason for vectors?

so fragments wont get digested once cleaved

What is some types of vectors?

plasmids and YACs (Yeast Artificial Chromosomes)

define plasmid

Usually circular double stranded DNA. Each has an origin of replication, several unique sites for restriction enzymes and a gene referred to as a selectable marker.
- typically 2-4 kb in size with an ability to carry inserts up to 15 kb in length

define YACs (Yeast Artificial Chromosomes)

linear vectors built to mimic a yeast chromosome (including telomeres and centromeres)
-Yeast DNA allows YACs to segregate as if they were natural chromosomes in the yeast host. Can carry insert up to one million base pairs.

What are the 2 problems with vectors?

1. not all plasmids will have an insert (& resultantly will self-ligate)
2. Not all cells will take up a plasmid. (With or Without insert)

how do you distinguish cells without plasmids or without inserts?

no inserts = lacz gene remains intact THEREFORE X-gal THEREFORE blue color on agar

if inserts present = lac z gene is disrupted THEREFORE no blue color.

Ampicillin is the in agar, making it turn blue or not

How do you retrieve the cloned insert DNA from purified recombinant plasmids?

cutting out the coned segment from an electrophoresis gel

What is the genomic library?

a collection of DNA clones that THEORECTICALLY contains copies of every DNA fragment in the whole genome inserted into a suitable vector and placed into storage.

What is the cDNA library?

first of all, cDNA is complementary DNA.
This library stores DNA sequences that have been copied from mRNA transcripts
-THESE sequences do not contain the intron sequences present in DNA
-they only contain the EXON sequences that actually code for the protein product

What are the differences between the Genomic and cDNA Libraries?

Genomic =
-contain all sequences
-contain same information no matter what cell type from the organism is used to construct the library
-used for prokaryotes mostly (no introns)

cDNA =
- contains only exon sequences
-information is conditional upon cell type ***
-used for eukaryotes mostly (to study expressed sequences)

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