Dna Manipulation Techniques (Chapters 3 & 4)

2. PCR (Polymerase Chain Reaction)

3. Restriction digest and ligation

4. Transformation

1. Agarose gel electrophoresis

2. Sequencing

1. Separate parental DNA strands

2. Primer for DNA polymerase to initiate synthesis

3. Synthesize DNA

4 and 5. RNA primer must be removed, bases filled, and gap sealed

Template DNA that needs to be replicated

Use high heat (95 C)

We can use DNA primer (oligonucleotide) that is synthesized in the lab

Taq DNA polymerase

DNA nucleotides (dNTPs)

MgCl (DNA polymerase requires Mg2+ to function)

It is able to remain intact and functional during the denaturing step (95 F), when most proteins denature at that temperature

These steps do not apply for PCR

They must all be added in order to maintain proper pH for optimal enzyme activity and stabilize DNA

1. DNA template

2. High heat to denature DNA and separate dsDNA to ssDNA

3. Primers

4. Heat stable DNA polymerase (Taq)

5. Deoxynucleotide triphosphates

6. Buffer containing Mg2+, among others

Genomic DNA extracted from cells

1. dATP

2. dGTP

3. dCTP

4. dTTP

1. Denaturation

2. Annealing

3. Extension

92 C - 95 C

55 C

72 C

dsDNA separates at high temperature to form ssDNA

Primers can base pair to ssDNA

Optimal temperature for heat stable DNA polymerases to work and a new strand is synthesized

To amplify the gene

Insert gene into vector in order to transport into cells

Verify gene is inserted into vector

Millions of copies of the gene are produced

Only amplify the gene of interest

A DNA vector

1 - 2000 kbases

False

True

False; covalently closed

True

True

1. Cleave 4 - 8 bp segment of dsDNA

2. Endonucleases

3. Palindromic recognition sequences

Sequences that are the same forwards as they are backwards

"race car"

"madam i'm adam"

1. Sticky ends

2. Blunt ends

1. Recognizes 5'-GATATC-3'

2. Complimentary sequence is implied

3. Cuts both strands after the 5'-T

4. Each fragment has blunt ends

1. Recognizes 5'-GAATTC3'

2. Complimentary sequence is implied

3. Cuts both strands after the 5'-G

4. Each fragment has sticky ends

Sticky ends have overhang and cut unevenly

Blunt ends do not have overhand and cut evenly

Restriction digest

From gel images

A methof of separating DNA fragments by size

Agarose

For florescent detection

Ethidium bromide

1. Separate dsDNA parental DNA

2. A primer is required for DNA polymerase to initiate synthesis

3. Synthesize DNA

4. The RNA primer must be removed and gaps must be filled in

5. Seal the backbone with final phosphodiester bond

1. Helicase --> separates strands

2. Gyrase --> assists

3. SSBs --> help stabilize

Primase

DNA polymerase III

1. Rnase H/DNA polymerase I

2. DNA polymerase III

Ligase

A technique that allows scientists to remove and/or replace genes with greater efficiency and accuracy than previous gene editing techniques

CRISPR/Cas 9

It was started China last year

To manipulate genes in a way to help treat cancer

The U.S.