Biochem Block 2: Recombinant Dna 1

Front 1

Restriction fragments are obtained by using...

Back 1

restriction endonucleases

Front 2

Restriction endonucleases cut the DNA in the (middle/end) of DNA at specific sites.

Back 2

-middle

Front 3

You need to use (the same/a different) restriction endonuclease to cut the starting DNA of interest and the vector in which you will put the DNA.

Back 3

the same so the DNA fragments can recognize each other

Front 4

Restriction endonucleases read _________, or adjacent sequences that are mirror images of one another.

Back 4

palindromes

Front 5

Can restriction enzymes work on single stranded DNA sequences?

Back 5

no. it only works on palindromes by looking at the top strand and the mirror image on the bottom strand...thus single stranded DNA has no palindromes and so restriction endonucleases cannot work

Front 6

Chimeric DNA is DNA made from...

Back 6

different origins (can put human DNA into a bacterial plasmid)

Front 7

What enzyme is used to join DNA fragments together?

Back 7

ligase

Front 8

Can you join a sticky DNA fragment with a blunt DNA fragment?

Back 8

No. must be complementary

Front 9

EcoR1 always gives you (blunt/sticky) ends.

Back 9

sticky

Front 10

How do you know that you successfully inserted a gene into a plasmid?

Back 10

for example: if you inserted a gene that gives a bacteria resistance to antibiotics, you must use a bacteria that is sensitive to that antibiotic. If the antibiotic resistance gene was successfully inserted, the bacteria will successfully grow onto the growth plate despite being in the presence of the antibiotic.

Also, you can put in a gene that creates a color along with the important gene you are inserting.

Front 11

What must you do to cause the bacteria take up a plasmid?

Back 11

shock the bacteria, making the membrane of the bacteria permeable

Front 12

Recombinant DNA can be used for all the following except:
1. identify for genes associated w/diseases
2. select for better intellectual performance
3. correct genetic defects
4. determine genetic differences between individuals

Back 12

2. select for better intellectual performance

Front 13

DNA produced from mRNA is called _______. This is done by the enzyme..

Back 13

cDNA ("DNA copy")

reverse transcriptase

Front 14

It is better to work with (DNA/RNA) because it is more resistant to destruction.

Back 14

DNA (double stranded while RNA is only single stranded)

Front 15

cDNA differs from DNA because it does not contain...

Back 15

introns (has already gone through splicing and all that because you are coming from mRNA)

Front 16

DNA sequences are synthesized with an automated technique called...

Back 16

PCR (polymerase chain reaction)

Front 17

Identifying DNA one nucleotide at a time is called

Back 17

DNA sequencing

Front 18

Why do you need a single stranded DNA or RNA to create a polynucleotide probe?

Back 18

so that it can anneal/hybridize with the specific sequence you want to tag with the probe

Front 19

You can make probes out of what 4 types of nucleic sequences?

Back 19

1. cDNA
2. genomic DNA
3. chemically synthesized oligonucleotide sequences
4. RNA (rarely because it is very sensitive to destruction)

Front 20

What are probes labeled with?

Back 20

32P...detected via autoradiography or fluorescence microscopy

Front 21

What do you do to double stranded DNA before it can be identified by a probe?

Back 21

treat it with heat or alkali so it becomes single stranded. then the complementary probe sequence with its attached 32P label can hybridize with the DNA strand

Front 22

With gel electrophoresis, DNA fragments are separated based on __________.

Back 22

size ONLY. not charge because DNA is all negative (bases and sugar are not charged--phosphate is negatively charged).

Front 23

In gel electrophoresis, DNA fragments migrate towards the ________.

Back 23

anode. despite its name, the anode is positively charged. just remember that anion goes to anode, cation goes to cathode.

Front 24

With gel electrophoresis, small DNA fragments migrate __________.

Back 24

the furthest

Front 25

With gel electrophoresis, we use ___________ gel to separate fragments with large size differences.

Back 25

agarose

Front 26

With gel electrophoresis, we use ___________ gel to separate fragments with few base differences

Back 26

polyacrylamide (the spaces in this gel are very tight)

Front 27

What chemical do we use to detect DNA fragments on a gel with UV light?

Back 27

ethidium bromide