Unit 3 AOS 1 Flashcards
Difference between DNA and RNA
- DNA is double stranded whilst RNA is single stranded.
- DNA has thymine whilst RNA has uracil.
- DNA also has deoxyribose sugar and RNA has ribose sugar
What are polymerase
Enzymes that catalyse the formation of polymers
What is the role of DNA polymerases
Unwinds the DNA double helix as a template to build the new DNA strands
Steps of DNA replication
- DNA is unwinded by the Helicase enzyme.
- Primer binds to the DNA template strand
- DNA polymerase attaches and moves along the strand adding bases from 5’ to 3’ ( left to right)
What is endonucleases
Also known as restriction enzymes. Range of enzymes responsible for cutting DNA
Restriction Enzymes
Target recognition site ( 4-6 bases )
How do restriction enzymes cut?
Cleave the phosphodiester bond of the sugar-phosphate backbone holding the DNA nucleotides together
Restriction Enzymes are also
A large group of enzymes that occur naturally in bacteria. They are a part of the bacteria’s cell defense system and target foreign DNA that may enter the cells such as DNA bacteriophages
Blunt End restriction enzymes
These guys leave clean cuts- cutting both sugar-phosphate backbone on BOTH STRANDS
Sticky end restriction enzymes
Cut DNA backbone at different places, leaving exposed bases for nucleotides
LIGASES
Are a group of enzymes that join fragments of DNA or RNA through ligation. This is the reverse of restriction enzyme’s roles
DNA ligase
Joins fragments of DNA. Join segments of newly replicated DNA and repairs breaks in DNA molecules. Also joins fragments from different organisms as long as they are cut with the same restriction enzyme
RNA ligase
Joins fragments of RNA
Ligation of Blunt ENDs
Is random, DNA ligase can join any 2 fragments together. Ligation blunt ends are more difficult to us in DNA manipulation processes that require the joining of specific frags
Ligation of sticky ends
Is SPECIFIC since the exposed bases are bound by complementary base pairing. Joins fragments by creating a phosphodiester bond between the 3’ OH and the 5’ phosphate end of the adjoining nucleotides. Also requires the fragments to be cut by the same restriction enzyme
PCR ( polymerase chain reaction)
Creates large quantities of identical DNA samples
What is used in PCR?
Targeted DNA to be amplified. Taq polymerase. Free nucleotides. Two DNA Primers
Why Taq polymerase used?
Because Taq polymerase is heat resistant and resists the changes in temp during PCR
What are Primers?
Single stranded RNA usually 30 bases long that are complementary to each end of DNA. Specifies start and finish of DNA frag that needs to be amplified
Steps in PCR?
- Denaturing: DNA sample heated to 95 degrees to break bonds b/w base pairs
- Annealing: Temp reduced to 50-60 degrees. Primers bind
- Extension: Temp increased to 72 degrees, Taq attaches each primer on the DNA strand and moves along each strand adding free nucleotides to form double stranded DNA.
DNA hybridisation
Measures the relatedness and its process includes: desaturation, hybridisation and melting
Gel Electrophoresis
Separates fragments of nucleic acids (DNA and RNA)
What charge is DNA?
DNA is negatively charged because of its phosphate Group
What gel is used?
Agarose gel
What are the terminals?
Where the loading wells are it is negative going to positive:
Neg -> pos
Large fragments and small fragments.
The smallest fragments travel the furthest and closest to the positive terminal. The larger fragments don’t travel very fast and are closer to to negative terminal
Genotyping
Genotyping uses gel electrophoresis to determine the genotype of an organism at particular allele
DNA profiling
Distinguishes between individuals on the basis of variable regions of their DNA
What are the variable regions?
They are non coding regions of DNA:
- Short tandem repeats (STR) ( 2-6 bases)
- Variable Number tandem repeats ( VNTR) ( 20-60 bases long)
Why aren’t coding regions used?
Coding regions are not useful as they code for the products/ functions that are the same for everyone