Lecture 15: DNA Manipulation - PCR and Molecular Clotting

What does polymerase chain reaction (PCR) do?

MAKES millions of copies of specific DNA sequence (in hours)

What principles does PCR use?

DNA Replication

What are the three steps of PCR?

Denaturation → Annealing → Extension

What happens during PCR denaturation?

• HEAT separates double-strand

• INTO two individual strands

What happens during PCR annealing?

• SOLUTION is cooled

• PRIMERS anneal (bind) to complementary DNA sequences

What bonds connect to the primer and DNA template strand?

Hydogen bonds

What are DNA primers called?

Oligonucleotides

What happens during PCR Extension?

• DNA polymerase extends primers to synthesize new DNA strands

• USES dNTPs

What are dNTPs a mix of?

dATP, dGTP, dCTP, dTTP

What are dNTPs?

Deoxyribonucleotide triphosphates

How many times is the PCR cycle repeated?

25 - 40 times

How many DNA copies are produced after each PCR cycle (equation)?

2ⁿ copies ( n = cycle number)

What happens after each PCR cycle (concept)?

DOUBLES DNA molecules

What is needed to design specific PCR primers?

DNA sequence information

What is Taq polymerase?

HEAT-STABLE DNA polymerase

Why is Taq polymerase used in PCR?

• SURVIVES heat used to separate DNA strand (doesn’t denature)

• ALLOWS repeated cycles of DNA replication

What enzyme uses an RNA template make a DNA?

Reverse Transcriptase

What is cDNA?

• COMPLEMENTARY DNA

• COPIED from RNA (using reverse transcriptase enzyme)

What is cDNA used for?

TEMPLATE for PCR

What type of enzyme is reverse transcriptase?

RNA-dependent DNA polymerase

Why can reverse transcriptase make an intronless copy of a gene?

USES mature mRNA (introns removd) to make cDNA

What is the process of using RNA template to make a DNA?

Reverse Transcription

What is the process (steps) to make intronless DNA copies?

mRNA → reverse transcriptase → cDNA → PCR → many DNA copies

What is telomerase (in terms of reverse transcription)?

• ENZYME

• HAS reverse transcriptase activity

• USES RNA template to extend chromosome (DNA) telomeres

What determines which DNA segment PCR will amplify?

• PRIMERS

• BINDS to complementary sequences flanking target segment

What are plasmids?

• SMALL, circular DNA molecules

• SEPARATE (distinct) from bacterial chromosomes

What are the three essential components of cloning a plasmid?

• Origin of replication (ORi)

• Antibiotic resistance gene (ex: AmpR, ampicillin resistance)

• Cloning site

How do plasmids amplify a gene of interest?

• BACTERIA replicates plasmids

• CONTAINS inserted gene

Where is the DNA of interest inserted into a plasmid?

MULTIPLE cloning site

What are restriction enzymes and what do they do?

• ENDONUCLEASES (enzyme)

• CUT plasmid and DNA of interest at specific sequences

What does DNA ligase do?

• JOINS DNA fragments from plasmid and DNA of interest

• SEALS backbone

What is recombinant DNA?

DNA formed by joining DNA from different sources

What is a restriction enzyme recognition site and what does it do there?

• SPECIFIC DNA sequence

• RECOGNIZES and cuts

What is a palindromic DNA sequence?

• READS sequence the exact same in both directions

• Example: (5’) GATATC (3’) = (3’) CTATAG (5’)

What are sticky ends?

• DNA ends with a single-strand overhanging

• Example:

5' - G     AATTC - 3'
3' - CTTAA     G - 5'

What are blunt ends?

• DNA ends with no overhangs

• Example:

5' - GAT | ATC - 3'
3' - CTA | TAG - 5'

How do sticky ends form?

RESTRICTION enzymes make staggered cuts in DNA

How do blunt ends form?

RESTRICTION enzymes cut straight across both DNA strands

How do restriction enzymes differ?

• HUNDREDS of restriction enzymes

• EACH recognizes and cuts at different DNA sequence

What does gel electrophoresis do?

SEPARATES DNA fragments by size

How is gel electrophoresis used in cloning?

• ISOLATES and extracts DNA fragments of interest

• SEPARATES fragments based on size

What Happens During Ligation?

• SAME restriction enzyme creates complementary sticky ends

• ON plasmid and DNA insert

• DNA ligase seals sugar-phosphate backbone

What is bacterial transformation?

• GENETIC alteration

• FROM uptake and expression of foreign genetic material

How do scientist help bacteria gain recombinant plasmids?

• INCREASE bacterial membrane permeability

• USES chemical treatment or electrical shock

How do scientists isolate bacteria with the new plasmid?

• ONLY few bacteria taken up by altered plasmid (transformation)

• GROW bacteria with ampicillin

• ONLY bacteria with antibiotic resistance gene survive

How do scientist select and grow bacteria with plasmids?

• PLATE bacteria on agar with antibiotic

• BACTERIA with resistance gene grows into colonies

• EACH colony has many bacterial cells containing markers (gene)

What is a selectable marker?

• GENE helping identify bacteria in plasmid

• Example: antibiotic-resistance gene

What is needed to express a human protein in E. coli?

• USING cloning vector (plasmid)

• NEEDS bacterial promoter, ribosome binding site, and transcription terminator

• CLONING intronless human cDNA into plasmids’s multiple cloning sites

What is the multiple cloning site

• REGION of plasmid

• GENE can be inserted using restriction enzyme