Molecular Genetic Analysis

quiz

Forward Genetics

Phenotype —> mutation

1) Mutants fly (with white eyes)

2) You discovers the gene responsible of red pigmentation

Reverse genetics

Mutation —> Phenotype

• A Wild type eyes fly with the gene x

• Create a nonsense mutations and see that it causes no pigmentation in the eye ( turns to white eyes

PCR

polymerase chain reaction

what is polymerase chain reaction (PCR) specifically used in lab experiment?

DnA synthesis in a test tube —> where they use this technique to make a lot of copies of a target sequences (DNA sequence on the tube that is used to copy) of DNA

What makes/brings(synthesis) the primer during DNA synthesis? What is needed to start (initiate) DNA synthesis? What moves along the strand that synthesis DNA?

• Primase

• Primer

• DNA polymerase

Where does DNA polymerase add dntps during replication?

• free 3’OH

Component of a PCR reaction (what is involved)

• Template DNA

• Taq polymerase

• dNTPS

• Primers

• Buffers

What does the template dna do in a PCR reaction

genomic extraction ( in a lab it is a process of releasing chromosomal DNA from inside cells and separating it from protiens, memebrane, and other material)

what does the taq polymerase do in a pcr reaction?

thermally stable Dna polymerase

what do dNTPS do in PCR reactions?

• deoxynucleotide thriphoasphates

• brings the building material for new DNA ( A’s, T’s, C’s, and G;s)

What do Primers (oligos) do in PCR reaction?

• small part within the sequence that determine the outer bounds of target sequence

• used initiate the reaction by providing 3’OH for taq polymerase

• used in pairs (forward and reverse primer)

what do buffers do in a PCR reaction? (dont need to know)

• maintain stable pH for the reaction

What term describes the growth of amplification during the cycle through 3 temperature in a PCR reaction?

Exponential amplification

Three stages of PCR reaction and their temps

1) 95 degrees - Denature

2) 55-65 degrees - Anneal

3) 72 degrees- elongation

What happens in the Denature stage of the PCR reaction?

Hydrogen bonds disrupted and created ssDNA

What happens in Anneal stage of the PCR reaction?

Primers anneal (bind from heating and cooling ) to target sequence

what happens in Elongation stage of the PCR reaction?

Traq polymerase synthesizes new DNA

how many cycles do samples typically cycle for? drntk

20-30

what is Gel electrophoresis/ what are they used for ?

used to separate molecules based on their size

Steps of gel electrophoresis

• nuclei acids are put into the gel matrix with a salt buffer

• an electrical charge is passed through the gel

• nucleic acids then travel to the positive pole

• label is added to DNA or gel (e.g. Etbr, racioactive label) that allows visualization

• sample is run alongs a size standard/ladder

Do smaller or larger fragments of nuleic acids run faster in the gel

smaller fragments

Restriction enzymes/endonuclease (cut nucleic acids inside dna/ rna strand) do

• isolate from bacteria (viral defense)

• cut dsDNA in sequence specific way

How long are recognition site (sites where proteins/Enzymes bind in DNA strand) for restriction enzymes

4 to 8 bp long

Palindromic ( dont need to know)

a nucleic acid sequence on double-stranded DNA where reading forward (5' to 3') on one strand matches the sequence reading forward on the complementary opposite strand

Most recognition sequences in restricted enzymes are…

palindromic

What are the two cuts that restriction enzymes make

1) staggered/ “sticky” ends

2 blunt ends

how do restriction enzymes recombine with DNA

• Cutting different sources of DNA with sticky end restriction will allow recombination

Vector

an agent that can carry DNA into a cell or organism

Examples of vectors

bacteriophages, viruses, plasmids

What is used to make copies of sequences in bacteria microorganisms

Cloning vector

The process of Plasmid cloning (type of cloning vector)

• Both the foregin DNA you want to ‘clone’ and the plasmid are cut with the same ‘sticky-end’ restriction enzyme

• recombined the cut sample of DNA and cut plasmid together w DNA ligase

what is bacterial transformation in plasmid cloning

is when the new foreign bacterial DNA from surrounding (target sequence) is in the plasmid it incorporates it

Typical Plasmind vector digram

MCS

• Multiple Cloning Site

• it is where your target sequence/ the foreign DNA goes