L14 DNA Sequencing and DNA Analysis

Question 1

What technique can be used to seperate DNA fragments base on their size?

Answer 1

Electrophoresis.

Question 2

What is the procedure of the mini-prep in sequencing?

Answer 2

1. Grow lots of bacteria.
2. Break them open.
3. Plasmids are small and compact
4. Genomic DNA is big and tangled and precipitates with otehr stuff.
5. Results in ug of purified DNA.

Question 3

Name few things that can go wrong during cloning?

Answer 3

• Empty vector
• Incorrect orientation
• Other weird stuff (occasionally: no plasmid, truncations)
• Often sequence to check for mutations – but need to know before this, whether it’s the right thing – screen several transformants

Question 4

What are the dyes used to visualize DNA and intercalates into DNA?

Answer 4

Ethidium bromide

Question 5

What is the basic method used for DNA analysis?

Answer 5

Using restriction enzyme digestion

Question 6

List the steps in the cloning startegy?

Answer 6

1. cloned insert using single RE sites.
2. Insert has other RE sites in it.
3. RE 2 and 3 will have different patterns.
4. 1 distinguishes between +/- insert
5. 2+3 will allow to asses the orientation.
6. Distinguish using gel electrophoresis.

Question 7

How do you do DNA analysis using PCR?

Answer 7

• Cloned insert using a single RE site (=1)
• Design some primers specific to vector and insert.
• Helps to add primers to products
• Will only get a PCR product with correct cloning product
• Sometimes presence/absence of a PCR product can be our diagnostic test (must have appropriate controls – here would have some other controls)
• Sometimes the size is what we are looking for
• Can also combine PCR with RE digest

Question 8

For what other analysing techniques can PCR be used?

Answer 8

1. Modern genetic fingerprinting.
2. Identification of repeat expansions.
3. Identification of genomic rearrangements.

Question 9

What does genetic fingerprinting do?

Answer 9

Amplifies regions that contain repeats.

Question 10

How do Variable Number Tandem Repeats (VNTRs) affect DNA fragment size?

Answer 10

The number of repeats in a VNTR region directly affects the size of the restriction fragment generated by an enzyme that cuts near the VNTR. More repeats lead to a larger fragment.

Question 11

How are VNTRs detected in DNA samples?

Answer 11

VNTRs are typically detected using Southern blotting. This technique involves:
1. Restriction enzyme digestion
2. Gel electrophoresis
3. Southern blotting: Fragments are transferred to a membrane.
4. Hybridization

Question 12

What can destroy a RE site?

Answer 12

Mutation in the sequence.

Question 13

What is the method used for DNA sequencing?

Answer 13

Sanger sequencing and chain termination method.

Question 14

What are some ways to check your DNA sequence?

Answer 14

• Cloning: Check for PCR-induced mutations.
• Sequencing: Compare your gene sequence to the wild type (WT) sequence.

Question 15

What are the two fundamental pieces of information required for DNA sequencing?

Answer 15

• Length of the DNA molecule: Knowing the number of base pairs.
• Identity of the last nucleotide: Determining the final base in the sequence.

Question 16

How does DNA sequencing work?

Answer 16

DNA sequencing is a specialized form of DNA synthesis.

• DNA is amplified to generate many copies of the target sequence.
• For each fragment: * The identity of the last nucleotide is determined. * The size of the DNA fragment is measured.

Question 17

How do dideoxynucleotides (ddNTPs) work in DNA sequencing?

Answer 17

• Difference at 3’ position - no OH group: ddNTPs lack the 3’ hydroxyl group present in normal deoxynucleotides (dNTPs).
• Termination of sequencing: This 3’ OH group is essential for forming the phosphodiester bond with the next incoming nucleotide. Without it, chain elongation stops.
• Identifying the last nucleotide: The type of ddNTP incorporated (ddATP, ddCTP, ddGTP, or ddTTP) reveals the identity of the last nucleotide in the growing DNA strand.

Question 18

How do you determine the identity of the last nucleotide in a DNA sequence using chain termination sequencing?

Answer 18

• Chain termination: The sequencing reaction is carried out in the presence of a dideoxynucleotide (ddNTP) that lacks a 3’ hydroxyl group.
• Specific termination: If ddATP is used, DNA synthesis will terminate specifically at every position where an adenine (A) would be incorporated.
• Fragment size analysis: Gel electrophoresis is used to separate the resulting DNA fragments by size. Each fragment represents a different termination point.
• Determining the last nucleotide: By analyzing the size of the fragments, the position of each adenine (A) in the original DNA sequence can be deduced.

Question 19

How do we know how long the DNA molecule is?

Answer 19

Gel electrophoresis – discriminate between DNA molecules by size

Question 20

If sequencing terminates at the first A (e.g.) how will we ever know the rest of the sequence?

Answer 20

We add both ddNTP and “normal” dNTPs.
Millions of products. Each will incorporate the ddNTP (and hence terminate) in a different place.

Question 21

How do you ensure a range of fragment lengths in chain termination sequencing?

Answer 21

• Problem: If sequencing terminates at the first occurrence of a specific nucleotide (e.g., A), you won’t obtain information about the rest of the sequence.
• Solution: The sequencing reaction includes both normal deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs) in a low concentration (typically 1:10 ratio).
• Result: This allows for both chain elongation and chain termination, generating a range of fragment lengths that can be used to reconstruct the entire DNA sequence.

Question 22

What was sanger sequencing originally like?

Answer 22

• Four separate reactions with different ddNTPs
• Radioactive labeling
• Gel electrophoresis
• Laborious

Question 23

What are the features of Modern Automated DNA Sequencing?

Answer 23

• One reaction contains all four ddNTPs - with different fluorescent labels
• Capillary gel electrophoresis - read by a computer
• Taq DNA polymerase - PCR-like reaction
• More sensitive and more specific
• Easy, quick and1 cheap!

Question 24

Name the components of a sequencing reaction?

Answer 24

• A DNA polymerase
• Deoxynucleotides (dNTPs)
• Dideoxynucleotides (ddNTPs) – fluorescently labelled
  ddATP, ddCTP, ddGTP, ddTTP
• Buffer (including Magnesium)
• Template DNA – plasmid / PCR product
• Primer