restriction enzymes

Restriction enzymes

Proteins that are used to cut DNA molecules into precisely sized fragments. They cut both the sugar phosphate backbone and hydrogen bonds between bases.

Exonucleases

Those enzymes that degrade DNA from the ends of the DNA strand

Restriction endonucleases

They break DNA molecules at internal positions

Types of cuts

staggered cut and clean cut

Staggered cut

exposes single stranded regions that are called "sticky ends"

Clean cut

Cut cleanly through the DNA molecule to produce "blunt ends"

Gel-electrophoresis

allows you to visualize and separate DNA ad proteins

CRISPR

Restriction enzyme

Genetic Engineering

Manipulation of DNA

Gel electrophoresis

The DNA will be separated into 'gelatin like" material using an "electric" current

Gel electrophoresis

DNA fragments will be separated according to their size and charge

DNA is negatively charged

When it's in an electrical field it moves toward the positive side

recombinant DNA

A DNA molecule carrying genes derived from two or more sources.

genetic engineering

The direct manipulation of genes for practical purposes.

restriction fragments

Molecules of DNA produced from a longer DNA molecule cut up by a restriction enzyme; used in genome mapping and other applications.

1st step of DNA fingerprinting

Make the agarose gel

2nd step of DNA fingerprinting

Extract DNA

3rd step of DNA fingerprinting

Cut DNA into tiny fragments using restriction enzymes

4th step of DNA fingerprinting

Insert DNA into wells of gel

5th step of DNA fingerprinting

Run a gel by electrophoresis to separate the pieces of DNA fragments by size

6th step of DNA fingerprinting

Stain the gel

7th step of DNA fingerprinting

Interpret the banding patterns

Unique DNA fragments of each person, these are also the differences in DNA b/n individuals

Restriction length polymorphism (RFLP's)

RFLP are the change in DNA sequence affects restriction enzyme's...

Cut site

Creates different fragment sizes and different band pattern

RFLP

Requirements for genetic engineering

Donor DNA, host bacteria, bacterial plasmid, and restriction enzymes

Another name for bacterial plasmids

extra chromosomal DNA

1st step of plasmid conjugation

cell to cell contact meditated by F-pili

2nd step of plasmid conjugation

Mobilization occurs by nicking the DNA at orit, rolling circle replication of f plasmid DNA and transfer of a single strand of DNA into the recipient cell

3rd step of plasmid conjugation

The second strand of DNA is synthesized in both the donor(leading strand synthesis) and recipient (lagging strand synthesis) cells. after synthesis is complete. the DNA is nicked at the nic site releasing the two DNA molecules the double stranded DNA circularizes in the recipient cell

4th step of plasmid conjugation

After the plasmid is completely transferred the cells separate. Both donor and recipient cells now have a complete f plasmid and can conjugate with other f minus recipient cells expression of surface exclusion proteins prevents mating between cells that contain plasmids.

What do plasmids do

antibiotic resistance and clean up oil spills by breaking down hydrocarbons

Applied transformations

most bacteria posses a genome composed of one double-stranded, circular DNA molecule, with all the genes necessary for survial

How many processes of gene transfer are there

three mechanisms

transduction

bacteriophage meditated. phage attach to recipient cell surface and inject entire genome into the lost cell

conjugation

plasmid meditated. only donor plasmid makes direct contact with host

transformation

when donor cell is lysed, DNA fragments released into surrounding. Host cells then incorporate fragments from medium

concentration of agarose is usally between

0.5% to 2.0%

what concentration of agarose did we use

0.8%

The higher the concentration....

the smaller the pore size making it easier to separate smaller fragments and they migrate faster

steps of gel- electrophoserisis

load wells, load tray, pour buffer, apply voltage, strain, destain, visualize and measure

mutation

any change in a DNA sequence away from normal.

what does a mutation imply

it implies that there is a normal allele that is prevalent in the population and that the mutation changes this to a rare and abnormal variant.

polymorphism

a DNA sequence variation that is common in the population.

what does a polymorphism imply

In this case no single allele is regarded as the strand sequence. Instead there are two or more equally acceptable alternitives

what dissolves blood clots in heart attacks

proteins

Electrophoresis uses what two aspects to separate DNA and RNA

Activated electrodes

Separation of macromolecules depends on two forces, what are they?

charge and mass

why is agarose used in gels

the concentration determines the size of the pores

What happens to negativity charged molecules

they migrate towards the positive pole

what happens to positively charged molecules

they will migrate toward the negative poles

Why did certain dyes migrate toward the positive electrode and others toward the negative electrode?

Because they have either a positive or negative charge and opposites attract

How does varying the concentration of agarose used in a gel affect the ability of the gel to separate molecules

Agarose gel with a lower concentration allows for larger molecules to move through it

Whats the same between chromotography and electrophoresis

both are separating methods and they both work with small particles

what can electrophoresis do

works with macromolecules and it can separate 100's to 1000's of molecules

what can chromotography do`

works with small molecules and it can separate about 20 molecules