SEQUENCING

CHAIN TERMINATION SEQUENCING

by Meet Patel

Introduction

(Image 1. sanger sequencing from the department of biological science.)

British Biochemist Frederick Sanger won two noble prizes, one for Insulin protein structure (in 1958) and the other for nucleotide sequencing (in 1980). In 1977, Sanger and colleagues have invented such a fabulous technique for determining the g sequence of DNA fragments or gene, which are known as Sanger sequencing. This method is based on chain termination PCR by modified nucleotide dideoxyribonucleoside triphosphates (ddNTPs). Thus the method becomes famous as chain termination sequencing. Now a day, several sequencing methods are available like automated sequencing and next-generation sequencing.

Principle

This method uses modified nucleotide ddNTPs, which are lack hydroxide group (-OH) at the 3’ position of deoxyribose sugar. Because of it, DNA polymerase cannot able to add the next nucleotide and produce various sizes of DNA fragments. Each ddNTPs are labelled with a specific fluorescence dye which is detected in the detecting machine (in automated method).

Methods

Basically in Sanger sequencing 3steps are main which are the following :

1. Chain termination PCR
2. Separate different sizes of DNA fragments on gel electrophoresis
3. Gel analysis/determination of sequence

 Let’s see all the steps in detail.

• Chain termination PCR: first of all we need to isolate purified DNA which is we want to sequence. Then amplify desired DNA fragments with conventional PCR. After the amplification step amplified fragments need to bind with adaptor (oligonucleotides) which are having a known sequence. So that we make a specific primer for chain termination PCR. This PCR is similar to convectional PCR but the difference is, in the chain termination PCR we need to use modified nucleotide, dideoxyribo nucleoside triphosphate, which is lacking hydroxide (-OH) group at 3’ position of deoxyribose sugar. All the PCR requirements add to the vial as well as add a small amount of ddNTPs. In the extension step, DNA polymerase recognizes both the nucleotides (DNA pol. cannot differentiate between dNTPs and ddNTPs.) and add them to the growing chain. When DNA polymerase adds ddNTPs rather than dNTPs, the growing chain terminated because of the lack of a 3’–OH group in the last nucleotide (DNA pol. need 3’-OH group for adding new nucleotide in growing strand.). The resulting product has many DNA fragments of various sizes.

(image 2. chain termination PCR. image from Pinterest.)

• In the manual method, four different vials are prepped for different ddNTPs. E.g. one vial has ddATPs, the second vial has GTP, the third vial has DDC tops, and the fourth vial has TTP. In each vial common terminated ends are present i.e. a vial containing ddATPs have all same nucleotide (Adenine) at the end of the strand.

(image 3. four vials have different ddNTPs in a different vial with another common PCR requirement. image from sciencemusicvideo.)

• In an automated method, one vial contains all of the ddNTPs and produce a mixer of fragment having all of the four nucleotides at the end of the strand, i.e. some strand has adenine, some strand has guanine, some strand has cytosine and the remaining strand have thiamine at the end of the strand. 

• Separation of different sizes of the fragment in gel electrophoresis: the resulting product of chain termination PCR is then added to the wells of the gel. The entire fragments are having the same net charge on them, so the separation accurses based on the size of the fragment. The smallest size of fragments move rapidly in the gel because of low resistance in migration and the largest molecules are move slowly due to high resistance in migration. DNA has a net negative charge because of the phosphate group in the backbone. So that they migrate towards the positive pole (anode). We all know that DNA polymerase adds nucleotides in the 5’ to 3’ direction so that the smallest bend reflects the first nucleotide in sequence and so on.

(image 4. gel electrophoresis in the manual method. in the automated method, capillary gel electrophoresis is used. image from Ask A Biology.)

• In the manual method, four different chain termination PCR products (each vial containing a different chain termination nucleotide in fragment with vary in size) are added in four different well (lanes) in the gel matrix and allowed it separate. In the automated method, capillary gel electrophoresis separates each DNA fragment based on its size. Because four different ddNTPs are labelled with four different fluorescent dyes, each fragment reflects different fluorescent color. 

•  Gel analysis/determination of sequence: at the extension process, if the DNA polymerase adds ddNTPs as the first nucleotide, the strand will not grow further and form the smallest fragment of DNA. If DNA polymerase adds ddNTPs as the second nucleotide, comparatively the fragment of DNA is large than the first one, and so on. DNA polymerase adds nucleotide in 5’ to 3’ direction. So in the manual method, the bend which is the last bend at bottom of the gel matrix is considered as the first nucleotide sequence, the second last consider as the second nucleotide sequence, and so on. Here basically two types of primer are used in the chain termination PCR process if the primer is comfortable for strand A (which are we want to sequence out.) the resulting sequence is complimentary with our desired sequence and one needs to convert in original (strand A) sequence. If the primer is comfortable for strand B (a complementary strand of desire sequence strand) the resulting sequence is correct of our desire i.e. no need to convert the n complementary strand.

( image 5. detector detects nucleotide sequence in an automated method. image from uoqasim.edu.iq.)

• In the automated method, all the fragments pass through capillary gel based on their size. At the end of the capillary, there are one laser device is present which give energy to the fluorescent labelled ddNTPs, which are labelled with a different fluorescent dye and produce different fluorescent color which is detected by CCD or other photosensitive camera and give a signal to the computer. This signal is converted in a graph known as chromatogram which gives us a correct sequence of DNA fragments.

 Application

1. To identify gene mutation which is causes genetic disorder(s).
2. Identify species by the reference sequence present in the gene database e.g. NCBI database.
3. Used in embryology technique i.e. IVF process to identify the zygote that has any mutation in the coding sequence.
4. Identify desired DNA fragment (gene) in plant recombination DNA technology.
5. Determined evolutionary relationship between two species.