genetic engineering

Question 1

siRNA?

Answer 1

small interfering RNA.

knockDOWN - used reduce the expression of a specific gene

Question 2

VNTR’s?

Answer 2

variable number of tandem repeats
short sequences of repeating nucleotide sequence
can be used to generate DNA profiling as vary in length in individuals

Question 3

transgenic mice ?

Answer 3

modify mouse ES cells by southern blotting or PCR
insert modified ES into host blastocyst
into another female - chimeric mice

Question 4

Knock out is mice

Answer 4

transgene (foreign DNA) injected into pronucleus of fertilised ova

Question 5

Yeast 2 hybrid screen

Answer 5

to find proteins thaat interact with your protein of interest

• create 2 hybrids 1. POI and DNA binding domain 2. POI and activation domain
• if POI’s are in close proximity they will bring there binding and activating domains together and form a transcription unit

Question 6

restriction fragment length polymorphism

Answer 6

a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases.

Question 7

RNA microarray

Answer 7

detect expression of thousands of genes at same time

• mRNAs converted to ssDNAusing reverse transcriptase
• appplied to slide
• ssDNA will hybridise to probe cell with complementary sequence

Question 8

immunohistochemistry and immunocytochemistry

Answer 8

histo- stains tissue sections
cyto - stains single layers of cells grown in culture

visualise location of a protein or antigen using antibodies

in situ

Question 9

DNA sequencing (sanger sequencing)

Answer 9

primer - 15-25 bases long
aneals to DNA sequence and is extended by DNA polymerase
terminator nucleotides incorporated - lack of OH on C3
add fluorescent tag

Question 10

restriction enzymes

Answer 10

isolated from bacteria as protect from bacteriophages
cut double stranded DNA, 4-8 bp leaving a palindromic sequence
can produce sticky ends

Question 11

western blotting

Answer 11

detect protein after separated by electrophoresis
primary antibody binds to antigen
secondary antibody binds to primary heavy chains and has a fluorescence
can see size and relative abundance of protein

in homogenate

Question 12

factors tha affect DNA migrations in electrophoresis

Answer 12

size - smaller DNA fragments move faster
Gel conc - DNA moves slowet in higher concentration
DNA shape- linear moves faster that circular and supercoiled faster than relaxed
gel type - agarose = 100 - 20,000bp
polyacrylamide = 10 -700 bps

Question 13

ways to lyse tissue to homogenise

Answer 13

mechanical - pestle and mortar
chemical - detergents
repeat freeze thaw

Question 14

northern blotting

Answer 14

visualisation and detection of RNA in homogenate

Question 15

southern blotting

Answer 15

visualisation and detection of DNA in homogenate

Question 16

chromosome painting

Answer 16

in situ detection of DNA - detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes

Question 17

cloning uses and types of organisms used as hosts

Answer 17

insert DNA into host = vector using restriction enzymes
host need short life span, easy cheap to grow, vast no. , easy to break up and purity DNA from

hence use bacteria and viruses

Question 18

DNA cloning process

Answer 18

• generate compatible cohesive ends on insert and plasmid by same enzyme
• fusion of insert using DNA ligase
• introduce recombinant DNA into bacteria
• antibiotic selction for bacteria containing

Question 19

plasmid ?

Answer 19

small circular DNA molecule within a bacteria that is physically separated from chromosomal DNA and can replicate independently.

• contains replication origin
• has antibiotic resistance gene to select

Question 20

2 types of DNA libraries

Answer 20

genomic - complete DNA sequence of organism

cDNA - derived from mRNA only represent expressed DNA

Question 21

PCR steps and uses

Answer 21

• DNA amplification
• 2 to the power number of cycles
• to detect infectious agents, forensics, modifications

1. template where start and end sequence known
2. put in thermal cylinder with Taq polymerase and dNTPS and primers
3. 95 decrees - denatures
4. 55 degrees annealing of primers
5. 72 degrees new strand synthesised