Lectures 1-2

Question 1

Nucleic Acid Hybridization

Answer 1

• Annealing of one strand to its compliment
• Can be used to find complements in a complex mixture
• Efficiency of hybridization can show how different the two strands are from one another

Question 2

Restriction Enzymes

Answer 2

In bacteria used to cut foreing DNA

These recognize palendrome

cut in squence specific manner–> this cut site can serve as a landmark

Question 3

Gel Electrophoresis

Answer 3

Seperate by size

Smaller peices move farther

Negative phosphate backbone allows for movement by voltage

Question 4

Sothern Blotting

Answer 4

Transfer from gel to paper

Dentature DNA with strng base

Probe paper for homology to sequence of interest

Question 5

Cloning

Answer 5

Isolation and amplification of specific DNA fragment

Restriction enzyme–> ID and isolate fragment of interest –> ligate this into cloning vector –> transform into bacteria –> Amplify

Question 6

Bacterial Plasmid

Answer 6

Small closed circle

Carry antibiotic resistance

Positive transformations are ID by plating

Question 7

Bacteriaphage lambda

Answer 7

double stranded linear DNA

Non essential regions of the genome are removed and replaced by DNA fragments (they contain genes for amplifying genome, protein coat synthesis and packaging DNA)

Question 8

Cosmids

Answer 8

Gitted version of phage

only keep area at end for replicating and packaging

this leaves more room for DNA

Question 9

Bacterial Artificial Chromosome (BACs)

Yeast Artificial Chromosomes (YACs)

Answer 9

Most useful and stable way to maintain a large peice of DNA

Can contain large peices of DNA but often undergoes recombination

Question 10

Genomic Libraries

Answer 10

isolated DNA of interest –> fragment it to controlled size –> random fragment ligation into vectors –> transformed into host –> replicated

Question 11

cDNA Library

Answer 11

Contruct library from mRNA

RNA isolated –> enrich it with poly A tail –> RT –> RNA degraded –> dsDNA formed –> ligate into vector

Abundance of clone can show expression pattern in specific tissue

Question 12

Screening Libraries

Answer 12

colonies lifted from petri dish to nitrocellulose paper

bacteria are lysed on paper then the DNA is denatured

DNA probes teh hybridize

Question 13

RT-PCR

Answer 13

Uses flouresence to quantify products at each step of the reaction

the resulting curve of product v. time can be used to compare template abundance in different samples

Question 14

Multi-plex PCR

Answer 14

A single sample is simultaneously analyzed with multiple sets of primers

Question 15

Use PCR to diagnose for presence of pathogens

(HIV)

Answer 15

Allows for detection even before infection manifest when it can most effectivly be treated

HIV: RNA RT–> DNA –> PCR

Question 16

Cloning by PCR

Answer 16

Isolate mRNA the amplify with PCR then clone with vectors

Question 17

Northern Assay

Answer 17

Enrich RNA by adding poly A tail

Seperate sample by Gel Electro.

transfer to nitro paper

then view

This can tell if mRNA for a gene is present and the expression level

Question 18

Protein Electrophoresis

Answer 18

Denatured: coat with neg charge so only seperating by size

Non-denatured: compare changes in charge between 2 proteins

Isoelectric focusing(IEF): seperate by charge only (pI)

2 dimensional: IEF then denatured

Question 19

Using antibodies to detect proteins

primary v. secondary antibodies

Answer 19

Primary: binds protein

Secondary: binds primary but is covalently tagged

location and abundance can be measured

Question 20

Western Assay

Answer 20

protein seperated by size with gel electro.

transfered to nitro paper

primary antibody

secondary antibody that is coupled to enzyme

incubate in a substrate for the enzyme

light is given off when enzyme is functioning

shows location and abundance of protein

Question 21

Immuniflourescence microscopy

Answer 21

Primary then secondary antibody(flourescent tag)

microscopy

tells if protein is expresesed and if it is in the proper location

Question 22

Micro- Arrays

Answer 22

used to simultaneously analyze the expression of thousands of genes

propes are short ssDNA complimentary to all predicted human mRNA

Question 23

Micro-Array type 1

Answer 23

isolate mRNA

make flouresenct labelled copy of mRNA

hybridized labelled coppy to micro-array

read signal

measures relative expressionlevel in a single sample

Question 24

Microarray type 2

Answer 24

RNA is isolated from refrence sample and labelled with color

RNA is isolated from sample and labelled with diff. color

they are simultaneously hybridized

ratio between two colors to compare relative expression

Question 25

Cluster Analysis

Answer 25

often MicroArray is used to find genes that are similarly expressed

and/or whose expression changes in a similar manner under different conditions

often genes dound in the same cludter frequently function in the samepathways or cellular process

Question 26

RNA Interference (RNAi)

Answer 26

mechanism used by cells to protect themselves against FNA viruses

Contruct gene that will express a self complimentary RNA

introduce it into the cell

This dsRNA is recognized by Dicer and helicase

dsRNA is cut into small pecies and then on of the strands is removed

this is used as a compliment to dins other mRNA in cell and destroy them