Biochemistry Test 2: Recombinant Methods

Front 1

Recombinant DNA

Back 1

Produced by splicing together 2 DNA molecules

Front 2

Reasons for recombination

Back 2

1. Clone large amounts of DNA
2. Genetically engineer cells to confer new characteristics
3. Produce products of spliced genes in bacteria
4. Genmod fruit
5. produce new organisms
6. stem cell research

Front 3

Restriction endonucleases

Back 3

Recognize specific palindromic sequences and can cleave strands in a way that leaves sticky ends for attachment of new strands

Front 4

Basic cloning strategy

Back 4

1. Anneal vector DNA with restriction frags
2. Propgate by transformation
3. select with antibiotics

Front 5

Eco R1 methylase

Back 5

Methylating palindromic sites can protect DNA from restriction endonucleases

Front 6

Good vector characteristics of plasmids

Back 6

Low weight
Many cloning sites
Origin of replication that allows self replication
High rate of replication

Front 7

4 common cloning vectors

Back 7

Plasmids
Bacteriophages
Cosmids
YACs

Front 8

pBR322

Back 8

Common plasmid vector
Has unique restriction sites
Has antibiotic resistance sites for selection
Has origin of replication (ori)

Front 9

YAC(yeast artificial chromosomes) and cloning

Back 9

Have ARS(autonomous replication sequences)

Front 10

Selection of clones

Back 10

After transforming, those cells that did not take up the plasmid are killed off with antibiotic. This is because the plasmids confer certain antibiotic resistance that the old cells did not hace

Front 11

cDNA formation(complementary DNA)

Back 11

cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the coding regions of an organism. While information in cDNA libraries is a powerful and useful tool since gene products are readily identified, the libraries lack information about enhancers, introns, and other regulatory elements found in a genomic DNA library.
This is made by cloning the mRNA, giving reverse transcriptase to make the DNA. Then erasing the mRNA with alkaline and synthesizing the full DNA.

Front 12

Genomic DNA library

Back 12

Contains the full complement of DNA of an organism

Front 13

Southern blotting

Back 13

Only gives info about the presence or absence of a gene and no infor about the expression.
DNA fingerprinting(RFLPs), genetic counseling, DNA mapping, sequence homology

Front 14

Northern blotting

Back 14

Fives info about RNA expression in a tissue. Only gives the info about one tissue

Front 15

Western blotting

Back 15

Fives info about expression of a given protein. We use antibody properties to separate these

Front 16

Expression vector

Back 16

A plasmid used to introduce and express a specific gene in a traget cell and protein. To detect them you use an antibody tag

Front 17

PCR

Back 17

Used to exponentially amplify DNA.
Denature the target DNA
Use Taq polymerase(heat stable) and dNTPs.
Heat and recool repeatedly to amplify DNA
The Taq extends the primers after each step

Front 18

Sickle cell

Back 18

Test with RFLP
Look for a Glu to Val substitution
The substitution cause a loss fo the MST 2 recognition site and the sickle cell DNA will be longer than normal
Sickle cell affects the beta subunit

Front 19

VNTRs

Back 19

Variable number tandem repeats
Baby Daddy probe
Also used for criminal ID

Front 20

Sanger Dideoxy sequencing

Back 20

Adding dideoxynucleotides to a sequence. Run PCR, see where the chain terminates and you can determine homology.

Front 21

Microarray

Back 21

Isolate 2 mRNA forms and reverse transcribe them into cDNA. Add fluorescent dNTPs. Then add to a microarray and the cDNA will anneal to complentary DNA and shine

Front 22

siRNA and gene silencing

Back 22

siRNA can specifically silence a mRNA being overexpressed which is a useful tool in diseases.